Polypeptide-induced monoclonal receptors to protein ligands

ABSTRACT

Monoclonal receptors raised to immunogenic polypeptides whose amino acid residue sequences correspond to sequences of oncoprotein ligands are disclosed, as are method for the production of those receptors and products and methods that utilize them. The monoclonal receptors bind both to the oncoprotein ligand to a portion of which the polypeptide corresponds in sequence, and to the immunogenic polypeptide to which the receptors were raised.

CROSS REFERENCE TO RELATED APPLICATION

This is a division of application Ser. No. 08/300,068, filed Sep. 2,1994, now U.S. Pat. No. 5,786,178, which is a file wrapper continuationof 07/772,702 filed on Oct. 7, 1991, now abandoned which is acontinuation of 06/736,545 filed on May 21, 1985, now abandoned, whichis a continuation in part of 06/702,954 filed on Feb. 15, 1985, now U.S.Pat. No. 5,030,656, which is a continuation in part of PCT applicationPCT/US84/01304 filed on Aug. 17, 1984. The totality of theabove-identified applications are incorporated by reference herein.

TECHNICAL FIELD

The present invention relates to immunological receptors and ligands,and more particularly to monoclonal receptors raised to polypeptideswhose amino acid residue sequences correspond to sequences of retroviraloncoprotein ligands.

BACKGROUND ART

Retroviruses are viruses that contain a single strand of RNA as thegenetic material rather than DNA. The single-stranded RNA genome of eachof these viruses gives rise to a double-stranded DNA molecule after thevirus infects a susceptible host. This DNA replica of the viral genomethen introduces itself permanently into a chromosome of the successfullyinfected cell and replicates in that host chromosome.

The retroviruses discussed hereinafter and in the claims may be furtherdefined as being replication-defective retroviruses. Thus, these virusesdo not themselves contain a gene encoding the reverse transcriptaseusually required to permit the viral RNA genome to be translated into aDNA that can be introduced into a chromosome of the infected host.Rather, the retroviruses discussed hereinafter typically must becomplimented in their infection by a so-called helper virus that isreplication-competent. That second virus contains the gene that encodesthe reverse transcriptase enzyme that incorporates the genomic materialsfrom both viruses into the successfully infected host cells to transformthose cells.

For ease in understanding, the replication-defective retroviruses willbe discussed hereinafter and in the claims merely as retroviruses withthe understanding that they are replication-defective and require theassistance of a helper virus for successful infection and transformationof host cells. This usage of the term retrovirus is known in the art andhas been used in the art as such without further explanation.

Some members of the retrovirus family are highly oncogenic as judged bytheir ability to cause the formation of solid tumors within a shortperiod of time after being inoculated into the host. These viruses canalso cause "cancerous" changes in cells grown and cultured in thelaboratory; such changes are called "transformations" and provide areliable in vitro biological assay for oncogenic viruses. Several suchviruses have been isolated from chickens, turkeys, mice, rats, cats andmonkeys.

A single gene, the oncogene, located on the genome of these highlyoncogenic viruses is responsible for the tumorgenic potential of thevirus. In the case of several viruses, the protein products of theironcogenes, referred to herein as oncoproteins, have been immunologicallyidentified by taking advantage of the fact that serum from an animalbearing a virus-induced tumor contains antibodies directed against thoseoncoproteins.

A rapidly growing body of evidence indicates that the oncogenes ofretroviruses are closely related to and are derived from specificgenetic loci in the normal cellular genetic information of allvertebrates. Molecular hybridization studies using specific nucleic acidprobes done during the middle 1970's, followed by genetic cloning ofviral oncogenes and their cellular relatives by recombindant DNAtechnology, have established the kinship between retroviral oncogenes(v-onc) and cellular oncogenes (c-onc) found in all normal vertebratecells.

Molecular analysis of the nearly two dozen retroviruses thus farisolated has revealed more than a dozen different oncogenes, eachdistinguished by its nucleotide sequence, and each with a correspondingcellular oncogenic homolog. For example, the human EJ or T24 bladdercarcinoma oncogene was identified as the homolog of the transforminggene of Harvey murine sarcoma virus (ras^(Ha)) and also of the BALBsarcoma virus (bas) [Parada et al., Nature, 297, 474-478 (1982); Der etal., Proc. Natl. Acad. Sci. USA, 79, 3627-3634 (1982); and Santos etal., Nature, 298, 343-347 (1982)]. In addition, the oncogene of thehuman carcinoma cell line LX-1 was found to be homologous to thetransforming gene of Kirsten strain of murine sarcoma virus (ras^(Ki))[Der et al., above]. Still further, the same v-onc for a c-oncdesignated fps of avian origin is represented at least twice among alimited number of avian retrovirus isolates; its mammalian cognatedesignated fes in feline species is found in two different strains offeline sarcoma viruses. Moreover, recent work has found a sequencehomology between human platelet-derived growth factor (PDGF) and theoncoprotein encoded by the simian sarcoma oncogene, v-sis, anddenominated p28^(sis) [Antoniades et al., Science, 220, 963-965 (1983)and Devare et al., Proc. Natl. Acad. Sci. USA, 80, 731-735 (1983)].

The structural and immunological relatedness between the transformingsis gene product (p28^(sis)) of simian. sarcoma virus and plateletderived growth factor (PDGF) provides the most solid link between thetransforming properties of oncogenes and the mitogenic action of growthfactors. The sis gene is one of many oncogenes that have been transducedat least in part by retroviruses. These captured genes have been highlyconserved through evolution, suggesting they serve importantphysiological functions. PDGF is a very potent mitogen for manyconnective tissue cell types in culture. It is stored in the alphagranules of platelets, and is released at sites of vascular damage. PDGFbinding to specific cell surface receptors triggering atyrosine-specific protein kinase activity. This event identifies acommon mechanism used by a wide variety of growth factors and oncogenes.

Insulin growth factor (IGF), gastrin, epidermal growth factor (EGF) andtransforming growth factors all bind to receptors that are associatedwith tryosine protein kinase activity. The oncogenes src, yes, fes, fps,ros, abl, fqr have tyrosine kinase activity while the oncogenes mos,raf, mht, and erb-B have sequence homology to kinase domain.Furthermore, sequence analysis of fragments of the EGF receptordemonstrate a very close homology with the predicted sequence of erb-B.Thus, the binding of a growth factor to a receptor with tyrosine kinaseactivity appears to be a common event in mitogenesis and transformation.

The precise molecular mechanisms of this interaction are not known. PDGFisolated from platelets contains two polypeptide chains that formdisulfide bonded complexes that migrate on denaturing polyacrylamidecgels between 20,000 to 35,000 daltons. Reduction destroys thebiological activity of these complexes and produces proteins thatmigrate between 14,000 and 18,000 daltons. Sequence analysis of thematerial migrating at 18,000 daltons identifies two homologous butdistinct sequences.

As discussed before, one of these sequences (PDGF-2) is highly homologusto the protein (p28^(sis)) predicted by the nucleotide sequence of thesimian sarcoma virus oncogene (sis). The homology begins at residue 67and extends at least to residue 171. Recently, the isolation andsequencing of a human c-sis clone has extended this homology to thepredicted carboxy-terminus. The open reading frame encoding thesequenced PDGF-2 region continues upstream, indicating PDGF isolatedfrom platelets is derived from a larger precursor, consistent with the4.2 kb sis-related mRNA detected in various cell lines. Cellular genesto which v-onc and c-onc genes and proteins are related are referred toas proto-oncogenes.

Another example of transduction of only a portion of a celluar gene by aretrovirus is the erb B oncogene. The erb B oncogene is highlyhomologous to a portion of the EGF receptor [Ullrich et al., Nature(London), 309, 418 (1984)]. Sequence analysis of the entire receptorgene demonstrates the relatedness of erb B with the entire intracellulardomain, the transmembrane domain, and a portion of the extracellulardomain.

Examples of additional oncogene products include those encoded by theN-ras oncogene that has extensive homology with both H-ras and K-ras[Taparowski et al., Cell, 34, 581 (1983); Dhar et al., Science, 217,934-937 (1982); and Tsuchid et al., Science, 217, 937-939 (1982)]; thePDGF-1 chain of platelet-derived growth factor that is homologous toPDGF-2, the polypeptide encoded by C-sis oncogene (above); and theinsulin receptor that is homologous to members of the src gene family[Ullrich et al., Nature (London) 313, 756 (1985)].

The protein encoded by the viral oncogene and having a corresponding,homologous protein within the host cell are both referred to herein asoncoproteins, although the cellular oncoprotein is typically larger andis present in small quantities in normal cells, and thus need not onlybe associated with neoplastic states. In addition, oncoproteins encodedby related oncogenes may have different molecular weights, e.g., the p85and p108 oncoproteins encoded by v-fes^(ST) and v-fes^(GA),respectively, and the 100-105 k dalton protein of normal mink cellsthought to be encoded by the c-fes gene. [Sen et al., Proc. Natl. Acad.Sci. USA, 80, 1246-1250 (1983).] The term oncoprotein is thus usedgenerally herein for proteins whose genes and amino acid residuesequences are homologous, at least in part.

The oncoprotein is generally not present in the virus particle thatinfects the cell, but is only expressed after infection andtransformation. The corresponding cellular oncoprotein is expressed atmost minimally in normal cells and to a greater extent in neoplasticcells. Thus, the oncoprotein cannot typically be obtained from thevirus. In addition, isolation of oncoproteins from cells is madedifficult because of the small amount present, the complex mixture ofproteins found in normal cells, and the relatively small amount of suchproteins present even in transformed cells.

Oncoproteins encoded by v-onc and c-onc genes thus typically containlarge sequences of amino acid residues that are homologous, butnevertheless are not usually identical. In addition, oncoproteinsencoded by genes of different viral strains, each of which containsostensibly the same oncogene, have been found to have slight variationsin their amino acid residue sequences as exemplified above, and by thefour published sequences of the ras gene which differ at the position ofthe twelfth amino acid residue. Thus, even when oncoproteins are inhand, it may be difficult to distinguish among them.

Immunologically induced receptor molecules such as monoclonal andpolyclonal antibodies or the idiotype-containing portions of thoseantibodies are useful in purifying protein ligands to which they bind,as diagnostic reagents for assaying the presence and quantity of theprotein ligands, as well as for distinguishing among homologous proteinligands.

The difficulties associated with obtaining quantities of oncoproteinstypically militate against the preparation of receptors to thoseoncoproteins, although whole cell-induced monoclonal antibodies to v-fesand v-fps encoded oncoprotein have been reported by Veronese et al., J.Virol., 43, 896-904 (1982). In addition, even were whole proteinsavailable for use as immunogens for inducing the production of suchreceptors, the use of large protein molecules as immunogens producesantisera containing polyclonal antibodies to the numerous epitopes ofthe large protein molecules.

Hybridoma and monoclonal antibody techniques utilizing whole proteins orlarge protein fragments as immunogens have been useful in narrowing theimmunological response to such immunogens. However, such technology asheretofore practiced has been extremely time consuming and has providedonly a relatively small number of hybridomas that secrete usefulantibodies that recognize the immunogen. Moreover, even when successful,such techniques cannot be predictive of the chemical identity of epitopeto which the receptor molecules are raised. Consequently, even afterimmunogen-recognizing receptors are produced, the obtaining of receptorsto specific, chemically identified epitopic portions of the proteinligand has been a hit or miss operation that still further reduces thenumber of useful hybridomas that are ultimately produced.

Arnheiter et al., Nature, 294, 278-280 (1981) reported on the productionof monoclonal antibodies that were raised to a polypeptide thatcontained 56 amino acid residues and corresponded in amino acid residuesequence to the carboxy-terminal portion of an intact interferonmolecule. That 56-mer polypeptide thus corresponded to approximatelyone-third of the sequence of the intact molecule.

Arnheiter et al. reported on the production of eleven monoclonalantibodies. However, only one of those eleven monoclonal antibodiesbound both to the polypeptide immunogen and also to the intactinterferon molecule. In addition, that binding was not very strong asjudged by the 3000-fold excess of intact interferon required to competethe antibody away from the synthetic polypeptide. None of the othermonoclonal antibodies bound to the intact molecule.

In addition, the production of the hybridomas secreting those monoclonalantibodies required the spleens from three immunized mice. The low yieldof the desired interferon-binding monoclonal antibodies, and the factthat three mouse spleens were needed for the preparation of thosehybridoma cell lines indicates that those workers were relativelyunsuccessful in their efforts.

Lerner et al. have been successful in obtaining protection of animals bythe use of vaccines against pathogens by utilizing synthetic amino acidresidue sequences of short to moderate length as immunogens. SeeSutcliffe et al., Science, 219, 495-497 (1983).

However, it must be understood that until the present invention,successful preparation of hybridomas and their secreted monoclonalreceptors differs from the successful preparation of a vaccinecontaining oligoclonal receptors. Thus, for a high yield monoclonalantibody preparation, it is necessary to stimulate B-cells to secretelarge amounts of avid antibodies. On the other hand, for a syntheticvaccine, a wider spectrum of oligoclonal antibodies may be produced insmaller amounts and with lower avidities. In addition, protection of ananimal against a pathogen typically requires both T-cell and B-cellactivations so that a cellular response and a humoral response,respectively, can be induced in the animal.

A popular explanation for the success of syntheticpolypeptide-containing vaccines in generating antibodies that recognizeintact proteins and protect animal hosts involves a stochastic model inwhich the diversity of the immune response allows the observation of aninfrequent event; i.e., the polypeptide adopting the confirmation of itscorresponding sequence in the native molecule. The concept thatmoderate-length polypeptides can frequently conform to native structuresis contrary to theoretical and experimental studies. Rather, suchpolypeptides are thought to exist as an ensemble of a large number oftransient conformational states that are in dynamic equilibrium. T-Cellactivation by, and B-cell production of antibodies raised to, some ofthat conformational ensemble have been believed sufficient to provideprotection upon vaccination.

BRIEF SUMMARY OF THE INVENTION

The present invention contemplates monoclonal receptor molecule thatbinds both (a) to a protein ligand encoded by a retrovirus gene, and (b)to a polypeptide of moderate length, about 7 to about 40 residues, andpreferably about 10 to about 30 amino acid residues, having an aminoacid residue sequence corresponding to an amino acid residue sequence ofa portion of the protein encoded by a gene of a retrovirus. The receptormolecule is raised to (induced by) an immunogen containing thepolypeptide. Most preferably, the receptor molecule is a monoclonalreceptor such as IgG or IgM class of immunoglobulins.

Specific, preferred monoclonal receptor molecules of this invention bindto proteins encoded by the genes listed below, and also to thepolypeptide(s) listed opposite those genes:

    ______________________________________                                        Gene       Polypeptide                                                        ______________________________________                                        fes        SDVWSFGILLWETFSLGASPYPNLSNQQTR;                                       -         SPYPNLSNQQTR;                                                       -        IHRDLAARNCLVTEKN;                                                    -       IGRGNFGEVFSG;                                                         -      LMEQCWAYEPGQRPSF;                                                      -     VPVKWTAPEALNYGR; and                                                    -    SSGSDVWSFGILLWE                                                          - myb    RRKVEQEGYPQESSKAG;                                                   -  RHYDEDPEKEKRIKELEL; and                                                    -  LGEHHCTPSPPVDHG;                                                           - sis      RKIEIVRKKPIFKKATV; and                                             -  RVTIRTVRVRRPPKGKHRKC;                                                      - ras      YREQIKRVKDSDDVPMVLVGNKC; and                                       -  KLVVVGAR(S, V, G)GVGK;                                                  ______________________________________                                    

wherein the amino acid residues in parentheses are each an alternativeto the immediately preceding amino acid residue in the formula;

    ______________________________________                                        myc         CDEENFYQQQQQSEL;                                                     -              PAPSEDIWKKFEL;                                                 -             LPTPPLSPSRRSGLC;                                                -            CDPDDETFIKNIIIQDC;                                               -           CSTSSLYLQDLSAAASEC;                                               -          CASQDSSAFSPSSDSLLSSTESSP; and                                      -         CTSPRSSDTEENVKRRT;                                                  - mos       LPRELSPSVDSR;                                                     -   RQASPPHIGGTY; and                                                         -  TTREVPYSGEPQ;                                                           B   ENDTLVRKYADANAVCQ;                                                           -      LGSGAFGTIYKG; and                                                      -     IMVKCWMIDADSRPKF;                                                       - PDGF-2   SLGSLTIAEPAMIAECKT;                                                -           RKIEIVRKKPIFKKATV;                                                -          RVTIRTVRVRRPPKGKHRKC;                                              - PDGF-1   SIEEAVPAVCKT;                                                      - abl          LMRACWQWNPSDRPSF;                                              - fms       FMQACWALEPTRRPTF;                                                 - src        LMCQCWRKDPEERPTF;                                                -            LGQGCFGEVWMG; and                                                -           CGSSKSKPKDPSQRRRS; and                                            - fgr      AMEQTWRLDPEERPTF.                                               ______________________________________                                    

The present invention also contemplates a method of producing monoclonalreceptor molecules to a protein molecule ligand. In this method, animmunogenic polypeptide of moderate length (about 7 to about 40residues), preferably synthetically produced, or a conjugate of thatpolypeptide bound to a carrier is provided. The amino acid residuesequence of that polypeptide corresponds to a portion of the amino acidresidue sequence of a protein ligand. That immunogenic polypeptide, whenbound as a conjugate to a carrier of keyhole limpet hemocyanin and usedto immunize a mouse, is sufficiently immunogenic and antigenic toprovide a 50 percent binding titer of the immunized mouse's serum to thepolypeptide of at least about a 1:400 dilution after threeimmunizations, each containing at least 10 micrograms of polypeptide inthe conjugate and using complete Freund's adjuvant for the firstimmunization and alum as adjuvant in the second and third immunizations.

A mammal is hyperimmunized with the immunogenic polypeptide or aconjugate of that polypeptide bound to a carrier to provide ahyperimmune serum that exhibits a 50 percent binding titer to thepolypeptide of at least about a 1:400 dilution. The receptor moleculesof that Serum also bind to the protein molecule ligand to which thepolypeptide corresponds in amino acid residue sequence.

The hyperimmunized mammal is maintained for a period of at least about30 days after the administration of the immunization that produces a 50percent binding titer of a dilution of at least about 1:400. A boosterimmunization, as by intravenous injection, is thereafter administered tothe animal.

Antibody-producing cells such as spleen cells (splenocytes) of theboosted mammal are fused with myeloma cells within a period of aboutthree to about five days from the. day of booster administration toprepare hybridoma cells. The hybridoma cells so prepared are assayed forthe production of monoclonal receptor molecules that bind to a proteinmolecule ligand to a portion of which the immunogenic polypeptidecorresponds in amino acid residue sequence. Preferably, the hybridomacells are also assayed for the production of monoclonal receptormolecules that bind to the polypeptide.

The hybridoma cells that produce monoclonal receptor molecules that bindto the protein molecule ligand are then cultured to prepare anadditional quantity of such cells. In preferred practice, thosehybridoma cells that are cultured are also those that produce monoclonalreceptors that bind to the polypeptide.

Another embodiment of the present invention contemplates a diagnosticsystem such as a kit for assaying for the presence of an oncoproteinligand. This system includes at least a first package containingmonoclonal receptor molecules of this invention. Admixing apredetermined amount of those receptors with a predetermined amount ofan aqueous composition to be assayed for the presence of an oncoproteinligand forms a receptor-ligand complex by an immunological reaction whenthe oncoprotein ligand includes an amino acid residue sequencecorresponding to the amino acid residue sequence of the polypeptidebound by the receptor molecule. The presence of the complex can beidentified by a label that is preferably contained in a second packageof the system. Preferred oncoprotein ligand-containing aqueouscomposition is amniotic fluid, urine, or concentrated urine. The urineor urine concentrate is easily obtained by noninvasive means and isreadily concentrated to allow the implementation of the diagnostic testset forth herein. Cell extracts and media conditioned by transformedcells are also suitable aqueous compositions containing oncoproteinligands.

An assay method is another contemplated embodiment of this invention.Here, a composition to be assayed for the presence of an oncoproteinligand such as serum, amniotic fluid, urine or a urine concentrate isadmixed with a liquid solution containing anti-oncoprotein receptormolecules. The admixture so formed is maintained for a period of timesufficient for a complex (immunocomplex; reaction product orimmunoreactant) to form between an oncoprotein ligand and receptormolecule (antigen-antibody complex). The presence of a complex isthereafter determined.

Where urine, as obtained or in concentrated form, is the composition tobe assayed, anti-oncoprotein receptors of any origin, e.g., polyclonal,oligoclonal or monoclonal, can be used in the instant invention. Themonoclonal antibodies of this invention are utilized with other samplesto be assayed. Determinations of the presence of an immunoreactant aretypically carried out using a radioisotope- or enzyme-labeled antibodyor Staphylococcus aureus protein A that binds to the receptor of theformed immunocomplex

A particularly novel aspect of this invention is the use of urine as abody sample. The assays described herein may be performed usingconcentrated urine as described, or may be performed using urine asobtained. oncogene-related proteins have not been heretofor identifiedin urine samples.

The assay aspects of this invention can be conducted using a pluralityof oncoprotein-related polypeptide ligands to provide a pattern ofimmunological reactivity for a particular assayed sample. Patternsobtained are compared to patterns obtained from individuals having knowndisease states to provide a diagnosis.

In yet another embodiment of this invention, monoclonal receptormolecules form the active, binding portions of an affinity-sorbantuseful for binding and purifying oncoprotein ligands. Here, thereceptors are linked to a solid support that is chemically inert to theoncoprotein such as agarose or cross-linked agarose. The affinitysorbant so prepared may then be admixed with an aqueous compositioncontaining a protein ligand to form a reversible receptor-ligand complexwhen the protein ligand has an amino acid residue sequencescorresponding to the amino acid residue sequence of the polypeptidebound by the receptor. The complex so formed can be thereafterdissociated to provide the protein ligand in a purified form.

The present invention provides several benefits and advantages.

One benefit of the invention is monoclonal receptor molecules that bindto epitopes contained in polypeptides of known amino acid residuesequence.

Another benefit of the invention is that monoclonal receptor moleculescan be raised that bind to epitopes contained in known amino acidresidue sequences of protein ligands encoded by retroviruses where thoseprotein ligands are not needed to induce the production of the receptormolecules.

One of the advantages of the present invention is the high yield methodof producing monoclonal receptors that bind to both an immunogenicpolypeptide of moderate length and to a protein ligand molecule to whoseamino acid residue sequence the polypeptide corresponds in part.

Another advantage of this invention is the provision of a diagnosticsystem such as a kit containing monoclonal receptor molecules capable ofassaying for the presence of an oncoprotein.

A further advantage of this invention is the provision of a diagnosticmethod that can be accomplished using body samples obtained bynon-invasive means.

Another advantage of this invention is that proteins of differingmolecular weights may be detected allowing a differential and highlyaccurate assessment of the precise oncogenes being expressed within theorganism.

A further advantage of this invention is the provision of a diagnosticmethod that allows prognostication of fetal development, or other growthstates including neoplasia that utilizes urine of the mother orindividual, respectively, in a non-invasive assay.

Still further benefits and advantages of the present invention will beapparent to those skilled in the art from the description and claimsthat follow.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings forming a part of this disclosure:

FIG. 1 is a photograph of an autoradiograph illustrating animmunological assay for detecting the presence of the ST-FeSV v-fesoncoprotein. Cell extracts from approximately 10⁵ MSTF cells, aproductively transformed mink cell line infected with Snyder-Theilenstrain of feline sarcoma virus (ST-FeSV) and feline leukemia virus-B(FeLV-B) [Sen et al., Proc. Natl. Acad. Sci. USA, 80, 1246-1250 (1983)],were electrophoresed onto a 5-17 percent polyacrylamide gel and thentransferred to nitrocellulose sheets. The transferred proteins were thenreacted with supernatants from hybridoma tissue cultures denominatedS10F03 (lane 1) or S22C06 (lane 2) or an anti-influenza hemagglutininhybridoma used as a negative control. This procedure of polyacrylamidegel separation follwed by transfer to nitrocellulose and visualizationis referred to hereinafter as a Western blot procedure. Proteinvisualization was accomplised as described in the Materials and Methodssection, hereinafter.

FIG. 2 is a photograph of an autoradiograph illustrating animmunological assay for detecting the presence of the FeSV fusionprotein denominated p85 (85 kilodaltons; 85 K daltons) by Western blotprocedures similar to those of FIG. 1. Cell extracts of approximately2×10⁶ MSTF cells were electrophoresed into a 5-17 percent polyacrylamidegel, and then electrophoretically transferred to nitrocellulose strips.The strips of nitrocellulose were incubated with 5 milliliters each ofhybridoma culture supernatant diluted 1:50 from hybridomas denominatedS10F03 (lane A); P43D09 (lane B); P42C10 (lane C); P44E11 (lane D); orwith R₂ 06B08, an anti-Rauscher gp70 protein receptor-producinghybridoma [Niman and Elder, Proc. Natl. Acad. Sci. USA, 77, 4524-4528(1980)], as a negative control (lane E).

Binding was visualized by addition of peroxidase-labeled rabbitanti-mouse IgG as is discussed in the Materials and Methods section,hereinafter. The marker "p85-" at the left side of FIG. 2 illustratesthe migration position of the 85 k dalton ST-FeSV polyprotein encoded bythe fes gene.

As can be seen from the proteins in lane E, this technique permitsvisualization of protein molecules that are not specifically bound bythe monoclonal receptors of this invention. Subtraction of thenon-specifically bound proteins visualized in lane E from the proteinsvisualized in lanes A-D illustrates that the only specifically boundprotein is the p85 oncoprotein encoded by v-fes.

FIG. 3 is a photograph of an autoradiograph illustrating animmunoprecipitation assay for the presence of the ³² P-labeled FeSVfusion protein denominated p85. CCL64 mink cells (MSTF cells; lanes Band D) or those infected with FeLV-B and FeSV (MSTF cells; lanes A andC) were each labeled for 2 hours with 1 microcurie of ³² P. The labeledcell extracts were then incubated with 5 microliters of goat anti-FeLVp15 antibodies (lanes A and B) or with 50 microliters of supernatantfrom cultured hybridoma S10F03 (lanes C and D). Immune complexes soprepared were collected using Staphylococcus aureus bacteria expressingprotein A. The precipitated complexes so collected were washed, and werethen dissociated into their component parts. The proteins werethereafter analyzed under reducing denaturing electrophoresis using a5-17 percent polyaccrylamide gel. The markers "p85-" and "pr65-" at theleft of FIG. 3 illustrate migration positions of the 85 K dakton ST-FeSVfusion protein encoded by the fes gene, and the 65 K dakton FeLVgag-precursor protein.

FIG. 4 is a graph illustrating immunoreactivities of oligoclonalantibodies raised to synthetic polypeptides corresponding in amino acidresidue sequence (i) to positions 139 through 155 of the predictedsequence of the simian sarcoma virus transforming protein denominatedp28^(sis) [Devare et al., Proc. Natl. Acad. Sci. USA, 80, 731-735(1983)] identified hereinafter as polypeptide (c) and as PDGF 2(73-89),and (ii) to residues 2 through 18 of the predicted amino acid residuesequence of the avian myeloblastosis virus oncoprotein [Rushlow et al.,Science, 216, 1421-1423 (1982)] identified hereinafter as polypeptide(d). The synthetic polypeptides conjugated to keyhole limpet hemocyanin(KLH) were used to immunize mice as is discussed generally in theMaterials and Methods section.

To test the specificity of oligoclonal antibody-containing sera soprepared, 250 nanograms of unconjugated polypeptide or 500 nanograms ofKLH were dried onto the bottoms of microtiter wells and fixed withmethanol as described by Niman and Elder, in Monoclonal Antibodies and TCell Products, Katz ed., CRC Press, Boca Raton, Florida, pp. 23-51(1982). The remaining portions of the wells were blocked againstnon-specific protein adsorption using 3% bovine serum albumin (BSA) anda 4 hour incubation period at 37 degrees C.

Into each well of the microtiter plate was instilled 25 microliters eachof two-fold dilutions of immunized mouse sera, starting with a dilutionof 1:400, using tissue culture medium supplemented with 10% fetal calfserum and were incubated with the BSA-blocked polypeptide or KLH for 16hours at 25 degrees C. After washing 10 times with distilled water, 25microliters of rabbit anti-mouse kappa antibody (Litton Bionics Inc.,Kensington, Md.) diluted 1:500 with 1% BSA in phosphate-buffered saline(PBS) were added and incubated for 2 hours at 37 degrees C. After anadditional 10 washings with distilled water, 25 microliters of goatanti-rabbit IgG conjugated to glucose oxidase and diluted 1:500 with 1%BSA in PBS were added and incubated for 1 hour at 37 degrees C.

The amount of glucose oxidase so bound was determined by addition of 50microliters of a solution containing 100 micrograms/milliliter of ABTSdye (Boehringer-Mannheim) in the presence of 1.2% glucose and 10micrograms/milliliter of horseradish peroxidase in 0.1 molar phosphatebuffer having a pH value of 6.0. The optical densities of the solutionsso prepared are read at 414 nanometers using a Titertech microscanner(Flow Laboratories Inc., Inglewood, Calif.).

Bindings exhibited by oligoclonal antibodies in sera raised to thesis-related and myb-related polypeptides are shown by open and closedsymbols, respectively. The antibody antigens are: sis-relatedpolypeptide (c) (, ∘); myb-related polypeptide (d) (▪, □); and KLH (♦,⋄).

FIG. 5 is a photograph of an autoradiograph illustrating animmunological assay for detecting the presence of non-reduced andreduced platelet-derived growth factor (PDGF) using mouse anti-seracontaining oligoclonal antibodies (receptors) induced by syntheticpolypeptides (c) and (d) as probes. PDGF extract was purified fromoutdated platelets as described in the Materials and Methods section.

Purified PDGF extract from approximately 2.5 units of platelets weremixed with a minimal volume of solution containing 0.5% sodium dodecylsulfate (SDS) and 5 percent of 2-mercaptoethanol. The resulting mixturewas boiled for 2 minutes and then electrophoresed therethrough a 5-17percent polyacrylamide gel. The protein was thereafterelectrophoretically transferred to nitrocellulose [Niman and Elder,Virology, 123, 187-205 (1982)] that was thereafter cut into strips,following the Western blot procedure.

The nitrocellulose strips so prepared were then treated with a solutioncontaining 3% BSA, 0.1% polyoxyethylene (9) octyl phenyl ether (Triton®X-100, Rohm and Haas Company, Philadlephia, Pa.) in PBS to inhibitnon-specific protein binding. 4 Milliliters of mouse anti-serum diluted1:200 were then incubated with the nitrocellulose strips.

After washing 3 times with a solution of 0.1% Triton® X-100 in PBS, thenitrocellulose strips were incubated either with 10⁶ counts per minuteof ¹²⁵ I-labeled Staphyloccous aureus protein A (lanes 2 and 3), or a1:1000 dilution of peroxidase-conjugated goat anti-mouse serum (Tago,Inc., Burlingame, Calif.), and again washed with 0.1% Triton® X-100 inPBS. The peroxidase conjugate was developed with a solution containing0.0009% H₂ O₂, 0.0025% 3,3'-dimethoxybenzidine dihydrochloride(Eastman-Kodak Co., Rochester, N.Y.) in a 10 millimolar Tris bufferhaving a pH value of 7.4. The ¹²⁵ I-labeled strips were developed byexposure on XRP-1 film (Eastman-Kodak Co., Rochester, N.Y.) using CronexHi-Plus (E. I. DuPont de Nemours & Co., Wilmington, Del.) intensifyingscreens at minus 70 degrees C. for 48 hours.

Lane 1 contains the total protein stained with amido black. The purifiedplatelet extract is shown probed with anti-sera raised to thesis-related polypeptide (c) (lanes 2 and 4) or the myb-relatedpolypeptide (d) (lane 3 and 5) as a negative control. External molecularweight standards based on BSA, ovalbumin, chymotrypsinogen andbeta-lactoglobulin are shown on the left.

FIG. 6 is a photograph of an autoradiograph illustrating an immunolgicalassay for the presence of PDGF following a Western blot proceduresimilar to that described hereinbefore. PDGF was boiled in the presence(lanes A-F) or absence (lanes G-L) of 10 percent 2-mercaptoethanol priorto electrophoretic protein separation, following the proceduresdescribed in Niman, Nature, 307, 180-183 (1984). Two oligoclonalantibody-containing antisera induced by the amino-terminal twelve aminoacid residues of PDGF-1 [denominated PDGF-1(1-12)] were used in lanes Aand G, and lanes B and H. Two oligonal antibody-containing antiserainduced by a polypeptide from a central portion of PDGF-2 [denominatedPDGF-2(73-89) and polypeptide (c)] that corresponds to the amino acidresidue sequence at positions 139 through 155 of p28^(sis) were used inlanes D and J, and in lanes E and K. Oligoclonal antibody-containingantisera induced by the amino-terminal seventeen residues of PDGF-2[denominated PDGF-2(1-17)] and by the twenty residues of PDGF-2 located36-16 residues from the carboxy-terminus [denominated PDGF-2(126-145)],corresponding to the sequence at positions 192 through 211 of p28^(sis),were used in lanes C and I, and lanes F and L, respectively. Antibodybinding to the proteins was visualized using rabbit anti-mouse IgG₁followed by 1.0⁶ cpm ¹²⁵ I-labeled Stachylococcus aureus protein A asdescribed in Niman, supra, and in the Materials and Methods sectionhereinafter.

FIG. 7 is a photograph of an autoradiograph illustrating animmunological assay for the presence of a 70,000 dalton protein in threecell lines using a Western blot procedure. An extract from approximately10⁶ cells per lane from each of SSV-transformed NIH 3T3 cells (lanesA-E), TRD1 cells (a spontaneously transformed Balb/3T3 cell line)(lanesF-J) and MSTF cells [a mink lung line (CCL64) productively infected withFeLV-B and the Snyder-Theilen strain of FeSVI (lanes K-O) wastransferred to nitrocellulose sheets following a Western blot procedure.Oligoclonal antibody-containing antisera induced by PDGF-1(1-12) wereused in lanes A-C, F-H and K-M. Oligoclonal antibody-containing antiserainduced by PDGF-2(73-89) were used in lanes D,E,I,J,N and 0. Theantisera were incubated with 100 micrograms of polypeptides PDGF-1(1-12)(lanes A,D,F,I,K and N), PDGF-2(1-17) (lanes B,G and L) andPDGF-2(73-89) (lanes C,E,H,J,M and O) prior to being immunoreacted withthe transferred cell extracts. Proteins were visualized as described forFIG. 6.

FIG. 8 is a photograph of an autoradiograph illustrating animmunological assay for the presence of p20^(sis) in culture mediaseparately conditioned by SSV-transformed normal rat kidney and normalrat kidney (NRK) cells.

Proteins from concentrated media, equivalent to 25 milliters ofnon-concentrated media, conditioned by SSV-transformed cells (lanesA,C,E and G) or NRK cells (lanes B,D,F and H) were separated andtransferred to nitrocellulose following the Western blot procedure. Thetransferred proteins were then admixed with oligoclonalantibody-containing antisera induced by PDGF-2(1-17) (lanes A-D) andPDGF-2(73-89) (lanes E-H). Sera were incubated with 100 micrograms ofpolypeptides PDGF-2(73-89) (lanes A,B,G and H) and PDGF-2(1-17) (lanesC,D,E and F) prior to being immunoreacted with the transferred proteins.Immunoreactions were visualized as described for FIG. 6. The marker"p20^(sis) " at the left side of FIG. 8 indicates the position ofp20^(sis).

FIG. 9 is a photograph of an autoradiograph illustrating animmunulogical assay for the presence of proteins encoded by or relatedto sis and fes antisera in urine from human cancer patients. The liquidbody sample in this assay was urine concentrate, obtained as describedin the Materials and Methods section. The concentrated urine waselectrophoresed into 5-17% polyacrylamide gel and then electrophoresedonto nitrocellulose.

Urine from three donors was concentrated 200-fold, dialyzed and 20microliters of each concentrate were electrophoresed and the proteinstherein transferred to nitrocellulose as described before. These threedonors had a rectal tumor (lanes A,D,G and J), a liver tumor (lane B,E,Hand K) and a Ewing's sarcoma (lanes C,F,I and L). An oligoclonalreceptor-containing antiserum induced by the sis-related polypeptidePDGF-2(73-89) that had been preincubated with the immunizing polypeptidewas used in lanes D-F, while the same antiserum that had beenpreincubated with the fes-related polypeptide corresponding to thesequence located at positions 744-759 of the v-fes^(ST) oncoprotein wasused in lanes A-C. Similarly, an oligoclonal receptor-containingantiserum induced by the above fes-related polypeptide that had beenpreincubated with the immunizing polypeptide was used in lanes G-I,while the same antiserum that had been preincubated with the abovesis-related polypeptide was used in lanes J-L. Immunoreaction (binding)between the oligoclonal receptors and the proteins was visualized asdescribed for FIG. 6. The positions of the sis- and fes-related proteinsdetected in the urine concentrates are indicated on the left and rightmargins by the markers "sis" and "fes", respectively.

FIG. 10 is a photograph of an autoradiograph illustrating animmunological assay for the presence of ras-related proteins in urine.

Urine was concentrated 250-fold (lanes A and B), 35-fold (lane C),70-fold (lane D), 75-fold (lane E) and 325-fold (lane F). The urine wasdialyzed, 20 microliters of each concentrate were electrophoresed andthe proteins therein were transferred to nitrocellulose as describedbefore.

The donors had been diagnosed as normal (lanes A, B and F), or as havingone of the following conditions: 38 weeks pregnant (lane C), lymphoma(lane D) and colon carcinoma (lane E). The same normal patient providedthe urine samples that were collected 14 days apart and were used inlanes A, B and F.

All urine samples were assayed using 10 microliters of anti-ras ascitesfluid incubated with residues 96-118 of the polypeptide p21^(ras) thathad been preincubated with residues 744-759 of the polypeptide fes^(ST)(lane A); residues 96-118 of the polypeptide ras^(Ha) (lane B); orresidues 139-155 of the polypeptide v-sis (lanes C-F). Immunoreaction(binding) between the oligoclonal receptors and the proteins wasvisualized as described for FIG. 6. The position of the ras-relatedproteins detected in the urine concentrates are indicated on the leftmargin by-the marker "ras".

The protein detected that is related to the ras oncogene is detected bya monoclonal antibody secreted by the hybridoma denominated ATCC No. HB8679 that was raised to a ras-related synthetic peptide. This protein ofapproximately 55 K daktons was detected in lane A and the activity wasblocked by a preincubation with the immunizing peptide (lane B). Urinecollected from the same normal individual contained the same protein twoweeks later (lane F). This protein has been detected in the urine of apregnant patient (lane C) and of a cancer patient (lane D and E).

FIG. 11 is a photograph of an auto-radiograph illustrating animmunological assay for the presence of a 23 K dakton protein in threecell lines using a Western blot procedure. The lanes of the Figure eachcontained an extract from about 10⁶ cells per lane from mink lung cellline transformed by the Snyder-Thielen strain of mink lung line sarcomavirus (MSTF) cells (lanes A-F) or from uninfected MSTF cell line CCL64(lanes G-L). The respective cell extracts were transferred frompolyacrylamide gel, onto nitrocellulose sheets, followed by a Westernblot procedure.

The extracts were assayed using antisera raised to a polypeptidecorresponding to residues 96-118 of p21^(ras) ("ras-1"; lanes A, B, GIH) that had been preincubated with a polypeptide corresponding toresidues 5-16 of v-ras^(HA) ("ras-2"; lanes A,G) or with a polypeptidecorresponding to residues 96-118 of p21^(ras) ("ras 1"; lanes B,H).

The same cell extracts were assayed with antisera raised to polypeptidescorresponding to residues 529-540 of p 85-fes ("fes-1"; lanes C,D,I,J)or to residues 744-759 of p 85-fes ("fes-2"; lanes E,F,K,L). Theantisera were preincubated with the fes-1 polypeptide (lanes D,J), withthe fes-2 polypeptide 744-759 (lanes F,L), or with the ras-1 polypeptide(lanes C,E,I,K) prior to being immunoreacted with the transferred cellextracts. Proteins were visualized as described for FIG. 6.

FIG. 12 is a photograph of an autoradiograph illustrating animmunological assay for the presence of a secreted protein insupernatants from spontaneously transformed mouse 3T3 cell line TRD-1(lanes A,B) or a human T-24 bladder carcinoma line (lanes C,D). Thesupernatants were assayed for presence of secreted fes-related protein.

The cell lines were grown in the absence of serum and collected after 48hours of growth. 35 Microliters of 1500:1 concentration of T-24 cellline supernatant or 1000:1 concentration of TRD-1 cells wereelectrophoresed into a polyacrylamide gel, and then transferred ontonitrocellulose.

Mouse antisera to v-fes^(ST) synthetic polypeptide corresponding toresidues ("fes-2") 744-759 of p85^(fes) ("fes-2") were utilized for theassay. The antisera were preincubated with a synthetic polypeptidecorresponding to residues 519-530 of v-fes^(ST) ("fes-1"; lanes A andB), or with the fes-2 polypeptide used to raise the antisera (lanes Band D).

The antisera were then immunoreacted with the transferred cellsupernatant. Proteins were visualized as described for FIG. 6.

FIG. 13 is a photograph of an autoradiograph illustrating animmunological assay for the presence of a ras related protein in a cellextract using a Western blot procedure.

A cell extract of approximately 10⁶ spontaneously transformed mouse 3T3cells was used in lanes A-D. 35 Microliters of a 1500-fold concentrationof 48 hour supernatants from mouse 3T3 TRD-1 cells were used in lanesE-H. The proteins of the supernatants were electrophoresed in apolyacrylamide gel, and then transferred onto nitrocellulose.

Oligoclonal antibody-containing antisera to v-ras^(HA) were preincubatedwith an unrelated fes polypeptide (lanes A,C,E,G) or the ras polypeptideused for the immunizations. (lanes B,D,F,H). Proteins were visualized asdescribed in FIG. 6.

FIG. 14 is a photograph of an auto-radiograph illustrating animmunological assay for the presence of a variety of proteins encoded byor related to sis, fes and ras oncogenes in urine using a Western blotprocedure similar to that described hereinbefore. The liquid body samplein this assay was urine concentrate, obtained as described in theMaterials and Methods section. The concentrated urine waselectrophoresed into 5-17% polyacrylamidize gel and then electrophroesedonto nitrocellulose.

Urine from 8 donors was concentrated 40-fold, dialized and 25microliters (the equivalent of 1 ml. of unconcentrated urine) waselectrophoresed and the proteins therein transferred to nitrocelluloseas described before. These donors had multiple myeloma (lane 1, Panels Aand B), gastric cancer (lane 2, Panels A and B; lane 1, Panels C and D),35 weeks pregnant (lane 3, Panels A and B), lymphoma (lane 4, Panels Aand B), gastric cancer (lane 1, Panels C and D), 36 weeks pregnant (lane2, Panels C and D), breast cancer (lane 3, Panels C and D), 39 weekspregnant (lane 4, Panels C and D and breast cancer (Panel E).

Monoclonal or oligoclonal receptor-containing antisera induced by sis-(Panels A and B), ras- (Panels C and D) or fes-related polypeptides(Panel E) were used to probe each sample to assay for the presence ofoncoproteins related to the immunizing polypeptides. Twenty microlitersof ascites fluid (induced by hybridoma ATCC HB 8679 and describedhereinafter, and induced by a hybridoma raised to the sis-relatedpolypeptide corresponding in sequence to positions 1-12 of PDGF-2;Panels A and B, and C and D, respectively) or mouse plasma [raised to apolypeptide corresponding in sequence to positions 744-759 of the fesoncoprotein (fes-2 polypeptide); Panel E] were preincubated for 30minutes at 37 degrees C with 100 micrograms of the immunizing raspolypeptide (Panels A, D and lane 2 of Panel E), sis polypeptide (PanelsB and C) or fes polypeptide (Panel E, lane 1), or with a polypeptidecorresponding to positions 366-381 encoded by erb B (Panel E, lane 3),or a polypeptide corresponding to positions 591-606 of ab1 (Panel E,lane 4).

Following preincubation, the samples were diluted 1 to 1000 in 3 percentBSA, 0.1 percent Triton® X-100 in PBS at a pH value of 7.4. The antiserawere then assayed as described hereinabove. Binding was visualized asdescribed in FIG. 6.

FIG. 15 is a photograph of an autoradiograph illustrating animmunological assay for the presence of ras and fes-related proteins inurine.

Urine was collected at monthly interval from a donor previouslydiagnosed as having active breast cancer (lanes 1, 4, 7, 2, 5, 8, 3, 6,9, Panel A). Urine was concentrated and dialyzed and an equivalent of 1ml unconcentrated urine was applied to each lane of Panel A.

In Panel B, aliquots of the same sample used in Panel A, lanes 3, 6 or 9was applied at the following equivalents of unconcentrated urine; 1000microliters (lane 1); 500 microliters (lane 2); 250 microliters (lane3); 125 microliters (lane 4); 60 microliters (lane 5); 30 microliters(lane 6); 15 microliters (lane 7); 7.5 microliters (lane 8).

The samples were prepared and probed with oligoclonal antisera toras-(positions 96-118; Panel A, lane 1-3); Panel B), fes-(positions759-769; Panel A, lanes 4-6) or sis-(PDGF-1 positions 1-12; Panel A,lanes 7-9) polypeptide as described for FIG. 14 except that nopreincubation with synthetic peptides was performed.

FIG. 16 is a photograph of an autoradiograph illustrating animmunological assay for the presence of ras- and fes-related proteins inurine. The donors of the assayed urine samples had been diagnosed ashaving recurrent breast cancer (lanes 1, 2) or were normal individuals(lanes 3-8).

The assay for ras-related proteins (Panel A) and fes-related proteins(Panel B) was conducted as described for FIG. 15. The samples assayedwere urine from a patient in clinical remission from breast cancer (lane1), the same patient 3 months later when the breast cancer reappeared(lane 2), and normal female (lanes 3-5) wherein samples were collected 3days apart, a normal female where samples were collected 12 hours apart(lanes 6-7) and a normal male (lane 8).

FIG. 17 is a photograph of an autoradiograph illustrating the detectionof sis- and ras-related proteins in urine samples from donors havingactive neoplastic disease. The urine was dialyzed and transferred tonitrocellulose and assayed with appropriate antisera as described forFIG. 15.

The donors had been diagnosed as having bladder cancer (lane 1),prostate cancer (lane 2), prostate nodule (lane 3) and lymphoma (lane4).

FIG. 18 is a photograph of an autoradiograph showing the detection ofsis-related proteins in urine samples from donors having cancer. Urinefrom donors with bladder cancer (lane 1), prostate cancer (lane 2),prostate nodule (lane 3), or lymphoma (lane 4) were prepared and probedwith antisera to sis (Panel A), ras (Panel B) or fes (Panel C) asdescribed in FIG. 15. The bands migrating slightly slower than p56^(sis)in lanes 1,2 represents excessive amounts of albumin in these samples.Although the increased levels of p56^(sis), p31^(sis), and p25^(sis)correlate with the increased albumin levels in Panel A, lanes 1,2, otherurine samples from donors with bladder or prostate cancer containedincreased levels of sis-related proteins in the absence of elevatedalbumin levels (data not shown). The slowest migrating bands in Panel B,lanes 1-3 identify p100^(ras) while the bands slightly faster than lightchain in Panel B lanes 1-4 identify p21^(ras).

FIG. 19 is a photograph of an autoradiograph illustrating the detectionof oncogene-related proteins in urine from a pregnant donor.

Four urine samples from the same individual collected at one weekintervals during the final month of pregnancy were probed with antiserato sis (PDGF-1 positions 1-12; Panel A), ras (positions 96-118; PanelB), or fes (positions 759-769; Panel C). Over exposure of Panel Cdemonstrates the presence of p35^(fes) (lanes 3 and 4) and p40^(fes)(lane 4). The protein migrating slightly faster than the light chainband (Panel C, lanes 1-4) or at the bottom of the gel (Panel C, lanes2-4) was detected with the mouse antisera to the fes peptide. Inaddition, a protein of 150,000 daltons was also detected with the mouseantisera to the fes peptide.

Urine samples were collected at one week intervals.

FIGS. 20, 21, and 22 are tables showing amino acid sequences of threeconserved regions of oncoproteins that have protein kinase activity.Those regions are denominated as "CONSERVED KINASE REGION" 1, 2 and 3,respectively, in FIGS. 20, 21 and 22. The oncogene encoding anoncoprotein having protein kinase activity is designated by its usualsymbol in the left-hand column. The middle column identifies thelocation in the oncoprotein polypeptide sequence, from theamino-terminus, of the conserved amino acid residue sequence. Theright-hand column shows the amino-acid residue sequences, from left toright and in the direction from amino-terminus to carboxy-terminus, ofthose conserved regions. The amino acid residue sequences are also thesequences of polypeptides useful as immunogens for inducing productionof the monoclonal receptors of this invention.

FIG. 23 is a table of data reflecting the detection of various levels ofthe oncogene-related proteins in 260 urine samples from pregnant donors.The samples were grouped according to the trimester of pregnancy.Multiple urine collections were obtained from any of the donors. Assayswere performed in accordance with the procedures and methods set fromhereinafter in the Materials and Methods section. As with the subset ofdonors having breast cancer, discussed hereinafter, very high levels ofp55^(ras) were detected in a group of pregnant donors thoughout thecourse of pregnancy. sis- and fes-related proteins increases as thepregnancy proceeds.

The levels of p55^(ras) changed dramatically in the course of several ofthe pregnancies. In contrast, levels of p55^(ras) detected in multiplesamples from normal or breast cancer donors, the concentration ofras-related proteins increased greater than 15-fold in one week incertain donors.

The concentration of the three sis-related proteins was approximatelythe same throughout the last month of pregnancy. p35^(fes) was detectedin the final two weeks of pregnancy while p40^(fes) was detected only inthe final week.

Urine samples taken six weeks postpartum continued to contain elevatedconcentrations of these sis-related proteins although the ras- andfes-related proteins returned to normal (data not shown).

FIG. 24 is a table showing the frequency of detection ofoncogene-related proteins in 51 control (normal donors) urine samplesand 189 urine samples from donors with a variety of mailgnancies. Theamount of oncogene-related proteins in the urine was estimated usingimmunoblots and placed into one of four categories; undetectable,detectable, 5 to 15-fold elevated and greater than 15-fold elevated. Theremaining types are listed as composite.

p21^(ras) was detected in approximately 70 percent of all samples fromdonors having neoplastic tumor disease. However, similar frequencieswere found in apparently normal individuals. The most striking elevationof p21^(ras) was detected in samples from donors having ovarian andgastric cancer as well as myeloma and molar pregnancies, all of whichhad greater than 15-fold elevations of this protein in at least 30percent of the samples.

DETAILED DESCRIPTION OF THE INVENTION

The present invention contemplates monoclonal receptor molecules tooncoprotein ligands, a general method of inducing or raising suchreceptors, and products and methods that utilize those receptors. Termsused frequently herein are defined as follows:

Receptor--A "receptor" is a biologically active molecule that binds to aligand. The receptor molecules of this invention are intact orsubstantially intact antibodies or idiotype-containing polyamideportions of antibodies. Biological activity of a receptor molecule isevidenced by the binding of the receptor to its antigenic ligand upontheir admixture in an aqueous medium, at least at physiological pHvalues and ionic strengths. Preferably, the receptors also bind to theantigenic ligand within a pH value range of about 5 to about 9, and ationic strengths such as that of distilled water to that of about onemolar sodium chloride.

Idiotype-containing polypeptide portions (antibody combining sites) ofantibodies are those portions of antibody molecules that include theidiotype and bind to the ligand, and include the Fab and F(ab')₂portions of the antibodies. Fab and F(ab')₂ portions of the antibodiesare well known in the art, and are prepared by the reaction of papainand pepsin, respectively, on substantially intact antibodies by methodsthat are well known. See for example, U.S. Pat. No. 4,342,566 toTheofilopolous and Dixon. Intact antibodies are preferred, and will beutilized as illustrative of the receptor molecules contemplated by thisinvention.

Monoclonal receptor--A "monoclonal receptor" (Mab) is a receptorproduced by clones of a single cell called a hybridoma that secretes butone kind of receptor molecule. The hybridoma cell is fused from anantibody-producing cell and a myeloma or other self-perpetuating cellline. Such receptors were first described by Kohler and Milstein,Nature, 256, 495-497 (1975), which description is incorporated byreference.

Oligoclonal receptor--An "oligoclonal receptor" is a receptor that isinduced by and binds to more than one epitope on a polypeptide ofmoderate length such as about 7 to about 40 or more preferably about 10to about 30 amino acid residues long. Oligoclonal receptors are usuallya mixture of receptors produced by more than one cell. Oligoclonalreceptors so produced are usually more epitopically specific in theirbinding than are the polyclonal receptors raised to whole proteinmolecules that can have epitopic regions throughout the length of theprotein chain or chains. Animals immunized with the polypeptides usefulherein produce sera containing oligoclonal receptors (antibodies).

Ligand--A "ligand" is the protein or polypeptide to which a receptor ofthis invention binds.

Corresponds--The term "corresponds" as used herein in conjunction withamino acid residue sequences means that the amino acid residue sequenceof a first polypeptide or protein is sufficiently similar to the aminoacid residue sequence contained in a second polypeptide or protein sothat receptors raised to the first (e.g., an antigenic syntheticpolypeptide) immunologically bind to the second (e.g., an oncoprotein)when the two are admixed in an aqueous composition.

The epitope-containing amino acid residue sequences of the correspondingfirst and second polypeptides or proteins are most preferably identical.However, changes, preferably conservative, in amino acid residues, anddeletions or additions of residues, within the epitope may be made and.still permit the cross-reaction of a receptor to the first polypeptideor protein with the second, as is known. Conservative changes in aminoacid residues are well known, and include exchanges of residues betweenlysine (Lys; K) and arginine (Arg; R), between aspartic acid (Asp; D)and glutamic acid (Glu; E), between leucine (Leu; L) and isoleucine(Ile; I) and the like.

The polypeptides useful herein are frequently described as having anamino acid residue sequence that corresponds to a portion of amino acidresidue sequence of a protein. Such polypeptides preferably only containamino acid residues that correspond identically, in addition to terminalresidues such as Cys residues utilized for binding or linking thepolypeptide to a carrier. Additional amino acid residues that do notcorrespond. to residues in the protein may also be present atpolypeptide terminii, but the use of such residues, while contemplatedherein, is usually wasteful, and is not preferred.

Similarly, proteins are described as having an amino acid residuesequence to a portion of which the amino acid residue sequence of apolypeptide corresponds. This terminology is intended to imply the samerelationship between the polypeptide and protein discussed hereinabove.

The full names for individual amino acid residues are sometimes usedherein as are the well-known three-letter abbreviations. The one-lettersymbols for amino acid residues are used most often. The Table ofCorrespondence, below, provides the full name as well as theabbreviations and symbols for each amino acid residue named herein.

    ______________________________________                                        Table of Correspondence                                                                             Three-letter                                                                            One-letter                                      Amino acid abbreviation symbol                                              ______________________________________                                        Alanine           Ala       A                                                   Arginine Arg R                                                                Asparagine Asn N                                                              Aspartic acid Asp D                                                           Asparagine + aspartic acid Asx B                                              Cysteine Cys C                                                                Glutamine Gln Q                                                               Glutamic acid Glu E                                                           Glutamine + glutamic acid Glx Z                                               Glycine Gly G                                                                 Histidine His H                                                               Isoleucine Ile I                                                              Leucine Leu L                                                                 Lysine Lys K                                                                  Methionine Met M                                                              Phenylalanine Phe F                                                           Proline Pro P                                                                 Serine Ser S                                                                  Threonine Thr T                                                               Tryptophan Trp W                                                              Tyrosine Tyr Y                                                                Valine Val V                                                                ______________________________________                                         (A. L. Lehninger, Biochemistry; Worth Publishers, Inc., N.Y., N.Y., 1970)

I. PRODUCTION OF MONOCLONAL RECEPTORS

As noted previously, the present invention contemplates monoclonalreceptor molecules that bind to an immunogenic polypeptide of moderatelength, e.g., about 7 to about 40 residues and preferably about 10 toabout 30 residues, as well as binding to a protein molecule ligand, aportion of whose amino acid residue sequence corresponds to the aminoacid residue sequence of that polypeptide. The monoclonal receptors ofthis invention are raised or induced by use of an immunogenicpolypeptide or conjugate of that polypeptide linked to a carrier; theimmunogenic polypeptide containing an amino acid residue sequence ofmoderate length corresponding to a portion of the amino acid residuesequence of the protein molecule ligand.

Epitopic localization of monoclonal antibodies poses technical problems.Monoclonal antibodies to the entire bacterial gene products can beproduced with two different types of immunogens, native or denatured.Use of native protein poses the most serious technical problemsregarding purification and subsequent epitope mapping. The chiefadvantage of using a native protein is the production of monoclonalantibodies that block the biological function of the target protein.

The oncogene product produced in bacteria is typically not structurallythe same as the gene product synthesized in higher organisms. Directevidence for this difference is provided by analysis of the sis geneproduct. In mammalian cells the p28^(sis) is rapidly cleaved intop20^(sis). In contrast, bacterial p28^(sis) s is not cleaved nor does itform a dimer.

Indirect evidence for differences between other oncogene productsproduced in bacteria or avian cells is provided by the observation thatmonoclonal antibodies raised against the E. coli-produced proteinproduct bind much more efficiently to the immunogen than to the proteinsynthesized in transformed chicken cells, even though the immunogen wasdenatured.

It is seen that the sequence of the viral oncogene can provide a basisfor identifying additional regions of a proto-oncogene sequence that canbe useful for synthesizing additional peptides for the generation andisolation of additional monoclonal receptors. Similarly, the sequenceanalysis of these proto-oncogenes identifies additional related peptidesthat have not yet been isolated in a retrovirus.

Thus, although purification of denatured protein is technically easier,the resulting antisera may recognize conformations unique to thebacterial gene product. This observation poses serious technicaldifficulties for epitope mapping studies.

Approaches for defining the epitope of the antibodies employ proteinfragments generated by partial proteolysis or expression of subgenomicfragments. Although mapping of epitopes using protein fragments wasfirst demonstrated by Niman and Elder, Proc. Natl Acad Sci USA, 77, 4524(1980), only an approximation of the binding sites could be made evenwhen several digests were assayed with a large panel of monoclonalantibodies. Thus, immunization even with protein fragments limits thedefinition of the binding site. Furthermore, there is no guarantee thatregions of interest will induce monoclonal antibodies.

In contrast, immunization with appropriate polypeptides of known aminoacid residue sequence as carried out herein, assures a production ofantibodies (receptors) that immunoreact with well defined regions; i.e.,regions that correspond to the sequences of the immunizing polypeptides.

Mapping of epitopes suggests that changing the epitope by one amino acidmay produce markedly different reactivities, while other studies showthat cross-reactivities are obtained when one or more amino acidresidues are different within the epitope. Furthermore, immunization ofthe same strain of mouse with the same synthetic polypeptide may producedifferent reactivities detected in the serum.

Hybridomas produced with synthetic polypeptides also produce monoclonalreceptors that react with the intact protein under a variety of reactionconditions because the recognition is largely conformationallyindependent. Therefore, Western blot, dot blot, fixed cells, and fixedtissues and body fluids such as amniotic fluid, and urine, eitherconcentrated or as obtained, can be assayed as well as native proteins.Furthermore, the known, precisely defined amino acid residues in theepitope allow isolation of antibodies that can distinguish single aminoacid changes, therefore providing a means of determining thesignificance of limited changes in conserved regions of relatedproteins.

Monoclonal antibodies against synthetic polypeptides also provide ameans of mapping sites of protein interaction. Differentialcoprecipitations of molecules associated with the pp60^(src) have beenreported, suggesting identification of regions of src proteins that areinvolved in such interactions.

Thus, inducing the production of monoclonal antibodies (receptors) withan immunogenic synthetic polypeptide assures isolation of antibodiesthat immunoreact with domains defined by the sequence of the immunizingpolypeptide does not require complex methodologies for isolation of thecorresponding immunogenic oncoprotein or the identification of thatoncoprotein's epitopic site, and produces receptors that recognize theoncogene product in a conformation independent manner, all of whichbroaden the application of such receptors for a variety of studies.

It was noted previously that although animal host protection has beenshown to be possible by the use of immunogenic polypeptides as theactive agents in vaccines, the ability to utilize such immunogenicpolypeptides to produce high yields of hybridoma tissue cultures thatsecrete avid monoclonal antibodies (Mabs) was not heretofore thought alikely possibility. Since each Mab is derived from a single cell thatproduces only one specificity, the ratio of the number of clonesproducing anti-polypeptide antibodies that also recognize the intactprotein molecule, to the total number of polypeptide recognizing clonescan provide a reasonable estimate of the true conformational frequencyof the polypeptide.

The results described herein are contrary to the before-mentionedstochastic model, and the frequency for the moderate-length polypeptidesused herein assuming a conformation similar to that of the nativeprotein is much higher than was previously expected. The frequency ofproducing hybridomas whose Mabs recognize both the synthetic polypeptideto which they were raised and the intact molecule is about 4 orders ofmagnitude (about 10,000) times greater than that predicted by thestochastic theory.

It is also noted that various workers have been utilizing immunogenicpolypeptides to raise antibodies that recognize those polypeptides forseveral decades. In addition, the above referenced Kohler and Milsteinarticle as to the production of monoclonal antibodies was published in1975. Since that date, 1975, Arnheiter et al., Nature (London), 294,278-280 (1981) described an attempt to prepare a monoclonal antibodyusing a polypeptide immunogen. As was previously noted, the Arnheiter etal. results must be viewed as a failure in that those authors requiredthe use of the spleens of three immunized mice and obtained only one IgGtype monoclonal antibody that recognized their large, 56-mer polypeptideas well as the protein to whose sequence that polypeptide corresponded.

It is believed that the relative paucity of published reports relatingto the preparation of monoclonal receptors prepared from immunogenicpolypeptides that recognize both the immunogen and a protein ligand towhose amino acid sequence the immunogenic polypeptide corresponds inpart is due to at least two factors. First, the prevalent thoughtfollowing the stochastic model predicts that few if any such monoclonalantibodies could be prepared. Second, the fact that workers such asArnheiter et al., above, did not possess a method suitable for thepreparation of the monoclonal receptors, inasmuch as the monoclonalreceptors of this invention that are raised to polypeptides are prepareddifferently from monoclonal antibodies prepared to whole proteins.

Thus, to successfully prepare Ig class monoclonal receptors thatrecognize both the immunogenic polypeptide and the protein ligand towhose amino acid residue sequence that polypeptide corresponds in part,one should follow the steps outlined hereinbelow.

An immunogenic polypeptide alone, or as a conjugate of that polypeptidebound (linked) to a carrier is provided. That polypeptide has an aminoacid residue sequence of moderate length, such as about 7 to about 40amino acid residues, and preferably about 10 to about 30 residues. Theamino acid residue sequence of the immunogenic polypeptide correspondsto a portion of the amino acid residue sequence of a protein moleculeligand such as an oncoprotein. While the immunogenic polypeptide can beused by itself as a ligand, it is preferred to use the polypeptideimmunogen as a conjugate bound to a carrier such as keyhole limpethemocyanin (KLH), albumins such as bovine serum albumin (BSA), humanserum albumin (HSA), red blood cells such as sheep erythrocytes, tetanustoxoid and edestin, as well as polyamino acids such as poly(D-lysine:D-glutamic acid), and the like.

The immunogenicity and antigenicity of the polypeptide may be tested bybinding the polypeptide to a keyhole limpet hemocyanin carrier as aconjugate, and then using the conjugate so prepared to immunize a mouse.The immunizing polypeptide or conjugate is dissolved or dispersed in aphysiologically tolerable diluent such as normal saline,phosphate-buffered saline or the like as are well known in the art. Anadjuvant, discussed below, is also included in the inoculum used forimmunizations.

A useful polypeptide is sufficiently immunogenic and antigenic toproduce a 50 percent binding titer of the immunized mouse's oligoclonalreceptor-containing anti-serum to the polypeptide that is at least abouta 1:400 dilution after three immunizations in a one-month period, eachof which immunizations contains at least about ten micrograms, andpreferably at least about 50 micrograms, of the polypeptide in theconjugate, and utilizing complete Freund's adjuvant for the firstimmunization and alum as adjuvant thereafter.

This test procedure need not be carried out prior to the use of a givenpolypeptide as immunogen, but it is preferable to do so as apre-screening technique to determine that polypeptides will be useful inpreparing the desired monoclonal receptors. Whether used as a pre-screenor not, the polypeptides useful herein as immunogens provide the abovetiter using the above immunization regimen.

Upon provision of the immunogenic polypeptide, a mammal such as a mouse,rabbit, goat, horse or the like, is hyperimmunized with the immunogenicpolypeptide or conjugate of that polypeptide bound to a carrier toprovide a hyperimmune serum whose receptor molecules exhibit a 50percent binding titer to the polypeptide of at least about a 1:400dilution. Thus, the same animal, e.g., a mouse, in which one may desireto pre-test the immunogenicity of the polypeptide may be used forraising the Mabs.

It is particularly preferred that the same animal that is used for apre-test be used for raising the Mabs. This preference stems from thefact that once the above 50 percent binding titer is achieved, thepreparation of hybridomas secreting monoclonal antibodies of the desiredspecificity using the spleen of that animal as the source ofantibody-producing cells is substantially assured, aside from theoccurrence of random laboratory mishaps such as contamination of cellcultures or otherwise destroying those cultures.

It is noted that the immunization regimen required to provide ahyperimmune state is a function, inter alia, of the animal type, animalweight, the immunogenicity and amounts of the polypeptide and carrier,if used, the adjuvant, if used and the number of immunizationsadministered in a given time period, as is known. The above-describedregimen for obtaining a 50 percent binding titer dilution of at leastabout 1:400 provides a hyperimmune state in the test mouse and may beused as a proportionalizable basis for inducing hyperimmune states inother animals. It is further noted that three immunizations are notnecessarily requited to provide the hyperimmunized state, but for auseful polypeptide, three such immunizations in a one-month period aresufficient to produce that state, or the polypeptide is not sufficientlyimmunogenic for the high yield production of hybridomas and theirmonoclonal antibodies of this invention.

The serum oligoclonal receptor molecules so produced in thehyperimmunized animal also bind to the protein molecule ligand, to aportion of which the immunogenic polypeptide corresponds in amino acidresidue sequence. Binding assays are described in the Materials andMethods Section hereinafter. It is noted that a pure sample of theprotein molecule ligand need not be utilized in these assays but rather,a cell extract or tissue preparation such as a microscope slidecontaining the protein ligand may be utilized.

The hyperimmunized animal is maintained; i.e., kept alive withoutadministration of further immunizations for a period of at least about30 days after administration of the immunization that produces a 50percent binding titer of at least a 1:400 dilution. In other words, theanimal is first immunized to provide a hyperimmunized state, and thenthe hyperimmunization is allowed to recede.

The decline in binding activity typically takes one to about five monthsfor mice. This decline in binding titer is believed to correspond to aperiod in which primed blast cells become capable of mounting a vigorousresponse when the immunogen is again introduced.

A booster immunization, as by intravenous injection, using theimmunogenic polypeptide or its conjugate is administered to the animalafter the period of maintenance is completed, e.g., at least 30 daysafter the last immunization. Antibody-producing cells, such as spleencells or lymph cells of the boosted animal are then fused with a myelomacell line from the same animal type (species) within a period of aboutthree to about five days from the day of booster administration toprepare hybridoma cells. The boost is believed to stimulate thematuration of the blast cells to the point at which those cells secretenearly optimal amounts of oligoclonal antibodies to the polypeptide.

The SP2/0-Ag14 (ATCC CRL 1581),hypoxanthine-aminopterin-thymidine(HAT)-sensitive, myeloma cell line ispreferred for use in fusion with mouse spleen cells, although other celllines such as P3X63-Ag8.653 may also be utilized. Details using this HATline for fusion are given hereinafter in the Materials and MethodsSection. The hybridoma cells are thereafter cloned at limiting dilutionfree from the presence of, or need for, feeder layers or macrophages toreduce overgrowth by non-producing cells, and to provide a selectionmethod for cells which grow readily under in vitro conditions. Suchfeeder layers may, however, be used.

The hybridoma cells so prepared are then assayed for the production(secretion) of monoclonal receptor molecules that bind to the proteinmolecule ligand. This ligand is a portion of the protein to which theimmunogenic polypeptide corresponds in amino acid residue sequence.Thereafter, the hybridoma cells that produce monoclonal receptormolecules that bind to the protein ligand are cultured further toprepare additional quantities of those hybridoma cells, and themonoclonal receptors secreted by those cells that bind to the proteinmolecule ligand. Typically, such culturing is done at limiting dilution,e.g., at an average of about one cell per culture-growing well.

In preferred practice, the hybridoma cells that are prepared are alsoassayed for the production of monoclonal receptor molecules that bind tothe polypeptide immunogen as well as to the protein ligand. Thereafter,hybridoma cells that produce monoclonal receptor molecules that bind toboth the immunogenic polypeptide and to the protein ligand are thosecells that are preferably cultured.

Where samples of the protein molecule ligand are limited, it isconvenient to first screen the hybridomas for secretion of monoclonalreceptors that bind to the immunogenic polypeptide. Hybridoma clonesthat exhibit positive binding to that polypeptide are then typicallyfrozen for storage. They are thereafter thawed, and subcloned bylimiting dilution for assurance that truly monoclonal antibodies areproduced, rather than a plurality of monoclonal receptors being producedfrom a plurality of different hybridoma cells. Those limiting dilutionsubcloning cultures are again typically carried out free from feederlayers or macrophages, as such are not necessary.

The hybridoma cells that are ultimately produced may be culturedfollowing usual in vitro tissue culture techniques for such cells as arewell known. More preferably, the hybridoma cells are cultured in animalsusing similarly well known techniques with the monoclonal receptorsbeing obtained from the ascites fluid so generated. The animals used forgeneration of the ascites fluid are typically 129xBALB/c mice bred inthe mouse colony of the Scripps Clinic and Research Foundation, LaJolla, Calif. However, when animals other than mice are used forpreparation of the hybridomas, that animal type is used for theproduction of ascites fluid.

As noted previously, it is preferred that the myeloma cell line be fromthe same species as the receptor. Therefore, fused hybrids such asmouse-mouse hybrids [Shulman et al., Nature, 276, 269 (1978)] or rat-rathybrids [Galfre et al., Nature, 277,131 (1979)] are typically utilized.However, some rat-mouse hybrids have also been successfully used informing hybridomas [Goding, "Production of Monoclonal Antibodies by CellFusion," in Antibody as a Tool, Marchalonis et al. eds., John Wiley &Sons Ltd., p. 273 (1982)]. Suitable myeloma lines for use in the presentinvention include MPC-11 (ATCC CRL 167), P3X63-Ag8.653 (ATCC CRL 1580),Sp2/O-Ag14 (ATCC CRL 1581), P3X63-Ag8U.1 (ATCC CRL 1597), andY3-Ag1.2.3. (deposited at Collection Nationale de Cultures deMicroorganisms, Paris, France, number I-078) and P3X63Ag8 (ATCC TIB 9).Myeloma lines Sp2/0-Ag14 and P3X63-Ag 8.653 are preferred for use in thepresent invention.

Thus, following the method of this invention it is now possible toproduce relatively high yields of monoclonal receptors that bind to orimmunoreact with known, predetermined epitopes of protein molecules suchas oncoproteins. In addition, once the skilled worker has producedhyperimmune serum containing oligoclonal antibodies that exhibit a 50percent binding titer of at least about 1:400 to the immunizingpolypeptide, that worker may follow the before-mentioned steps, take thespleen from the hyperimmunized animal, fuse its antibody-producing cellswith cells of a myeloma line from the same animal type or strain, and besubstantially assured that one or more hybridomas produced from thatfusion secrete monoclonal receptors that bind to the immunizingpolypeptide and to the corresponding protein, such as an oncoprotein.Such results were not heretofore possible.

The above method is useful for preparing hybridomas that secretemonoclonal receptors to substantially any protein molecule ligand.Illustrative of such hybridomas and their monoclonal receptors are thoseraised to immunogenic polypeptides of moderate length whose amino acidresidue sequences correspond to amino acid residue sequences ofoncoproteins encoded by oncogenes. Exemplary oncogenes and usefulimmunogenic polypeptides are shown below followed by the parenthesized,numerical position from the amino-terminus in the oncoprotein sequenceto which the polypeptide corresponds wherein the amino acid residuesequences of those polypeptides are given from left to right and in thedirection of amino-terminus to carboxy-terminus, and are represented bya formula selected from the group consisting of formulae shown in Table1, below:

                                      TABLE 1                                     __________________________________________________________________________    Oncogene  Polypeptide Sequence                                                __________________________________________________________________________    v-fes.sup.ST*                                                                           SDVWSFGILLWETFSLGASPYPNLSNQQTR (693-722);                              -                    IHRDLAARNCLVTEKN (632-647);                              -                   SSGSDVWSFGILLWE (690-704)                                 -                  IGRGNFGEVFSG (519-530);                                    -                 LMEQCWAYEPGQRPSF (744-759);                                 -                VPVKWTAPEALNYGR (674-688); and                               -               SPYPNLSNQQTR (711-722);                                       - v-myb*        RRKVEQEGYPQESSKAG (2-18); and                                 -              RHYTDEDPEKEKRIKELEL (94-112);                                  - v-sis*          RKIEIVRKKPIFKKATV (139-155); and                            -             RVTIRTVRVRRPPKGKHRKC (192-211);                                 - v-ras.sup.Ha*        YREQIKRVKDSDDVPMVLVGNKC (96-118); and                  -     KLWVGARGVGK  (5-16);                                                    - v-ras.sup.Ki*        KLVVVGASGVGK (5-16);                                   - T24-ras.sup.Hu*      KLVVVGAVGVGK (5-16);                                   - c-ras.sup.Hu*      KLVVVGAGGVGK (5-16);                                     - c-myc.sup.Hu*      CDEENFYQQQQQSEL (25-40);                                 -     PAPSEDIWKKFEL (43-55);                                                  -    LPTPPLSPSRRSGLC (56-70);                                                 -   CDPDDETFIKNIIIQDC (117-133);                                              -  CSTSSLYLQDLSAAASEC  (171-188);                                             -                  CASQDSSAFSPSSDSLLSSTESSP  (208-231);                       -                 APGKRSESGSPSAGGHSKPPHSPLVLKRC  (272-300);                   -                CTSPRSSDTEENVKRRT (342-358); and                             -                  AEEQKLISEEDLLRKRLRRQLKHKLEQLRNSCA  (408-439);                       - v-mos*        LPRELSPSVDSR (42-53);                                -             RQASPPHIGGTY (260-271); and                                     -            TTREVPYSGEPQ (301-312);                                       B*      ENDTLVRKYADANAVCQ (25-41);                                               -             LGSGAFGTIYKG (138-149); and                                     -            IMVKCWMIDADSRPKF (366-381);                                      - PDGF-2*       SLGSLTIAEPAMIAECKT (1-18);                                    -                  RKIEIVRKKPIFKKATV (73-89); and                             -                  RVTIRTVRVRRPPKGKHRKC (126-145);                            - PDGF-1*       SIEEAVPAECKTR (1-12).                                      __________________________________________________________________________     *v-fes.sup.ST = Polypeptides from predicted sequences encoded by the fes      oncogene of SnyderTheilen strain of feline sarcoma helper virus. Hampe et     al.y Cell, 30, 775785 (1982).                                                 vmyb = Polypeptides from predicted sequences encoded by the myb gene of       avian myeloblastosis virus. Rushlow et al., Science, 216, 14211423 (1982)     vsis  = Polypeptides from predicted sequences encoded by the sis gene of      simian sarcoma virus.  Devare et al., Proc. Natl. Acad. Sci. USA, 79,         31793182 (1982).                                                              vras.sup.Ha = Polypeptides from predicted sequences encoded by the ras        oncogene of Harvey murine sarcoma virus. Dhar et al., Science, 217, 93493     (1982).                                                                       vras.sup.Ki = Polypeptides from predicted sequences encoded by the ras        oncogene of Kirsten murine sarcoma virus.  Tsuchida et al., Science, 217,     937939 (1982).                                                                T24ras.sup.Hu = Polypeptides from predicted sequences encoded by the ras      oncogene of human bladder carcinoma. Reddy et al., Nature, 300, 149152        (1982).                                                                       cras.sup.Hu = Polypeptides from predicted sequences encoded by the ras        oncogene of normal human cells. Reddy et al., Nature, 300, 149152 (1982).     cmyc.sup.Hu = Polypeptides from predicted sequences encoded by the myc        oncogene of normal human cells. Colby et al., Nature, 301, 722725 (1983).     vmos = Polypeptides from predicted sequences encoded by the mos oncogene      of normal human cells. Van Beveren et al., Nature, 289, 258262 (1981).        verb  B = Polypeptides from predicted sequences encoded by the erb B          oncogene of avian erythroblastosis virus. Yamamoto et al., Cell, 35, 71       (1983).                                                                       PDGF2 = Polypeptide from sequence encoded by the gene for human               plateletderived growth factor, chain 2. Doolittle et al., Science, 220,       275277 (1983).                                                                PDGF1 = Polypeptide from sequence encoded by the gene for human               plateletderived growth factor, chain 1. Doolittle et al., Science, 220,       275277 (1983).                                                           

The homologous polypeptides encoded by the above four ras genes may beconveniently written as one amino acid residue sequence, from left toright and in the direction from amino-terminus to carboxy-terminus,represented by the formula

    KLVVVGAR(S,V,G)GVGK

wherein the amino acid residues in parentheses are each an alternativeto the immediately preceding amino acid residue, "R", in the formula.

Further immunogenic polypeptides useful for inducing the production ofmonoclonal receptors of this invention are shown in Table 2 below,wherein the oncogene abbreviations and parenthesized positions are asdescribed for Table 1.

                  TABLE 2                                                         ______________________________________                                        Oncogene  Sequence                                                            ______________________________________                                        sis       DPIPEELYEMLSDHSIRSF (8-26);                                            - v-ras.sup.Ki         YREQIKRVKDSEDVPMVLVGNKC (96-118);                      - v-ras.sup.Ha         YTLVREIRQHKLRKLNPPDESGPGC (157-181);                   - v-ras.sup.Ki         YTLVREIRQYRLKKISKEEKTPGC (157-180);                    - v-src             GSSKSKPKDPSQRRRS (2-17);                                  -                   LGQGCFGEVWMG(273-284); and                                -                  LMCQCWRKDPEERPTF (494-509);                                - v-myb              LGEHHCTPSPPVDHG (159-173).                            ______________________________________                                    

Still further useful polypeptides for inducing the production ofmonoclonal receptors of this invention are the polypeptides whoseoncogene, position in the oncoprotein sequence and polypeptide aminoacid residue sequences are shown in FIGS. 20, 21, and 22. Thosepolypeptides correspond to sequence-conserved regions in the well knownfamily of protein kinase oncoproteins, some of whose oncogenes have beenpreviously noted herein.

II. MONOCLONAL RECEPTORS

While the present invention contemplates a large number of monoclonalreceptors, five such receptors, intact monoclonal antibodies (Mabs),will be discussed in detail herein as illustrative of the group. Theabove-discussed test for the immunogenicity and antigenicity of thepolypeptide will be discussed thereafter for polypeptides correspondingto additional monoclonal receptors that bind to different oncoproteins.

A. Exemplary Receptors

Five exemplary monoclonal receptors were raised to the v-fes related,30-mer immunogenic, synthetic polypeptide shown below (polypeptide a),and each also binds to the carboxy-terminal 12-mer polypeptide shownbelow (polypeptide b), as well as binding to the oncoprotein denominatedp85 (85K daltons) encoded by the v-fes gene of ST-FeSV. The amino acidresidue sequences of synthetic polypeptides (a) and (b), from left toright and in the direction from amino-terminus to carboxy-terminus, arerepresented by the formulae

    ______________________________________                                        polypeptide a                                                                              SDVWSFGILLWETFSLGASPYPNLSNQQTR;                                  polypeptide b SPYPNLSNQOTR.                                                   ______________________________________                                    

The hybridomas secreting these Mabs were denominated S10F03, S22C06,P43D09, P42C10 and P44E11. Four of the above hybridomas were received atthe American Type Culture Collection (ATCC) of Rockville, Md. on Aug. 2,1984, and were given the designations ATCC HB 8596 (S10F03), ATCC HB8595 (S22C06), ATCC RB 8594 (P43D09), and ATCC HB 8593 (P44E11),respectively. Hybridoma HB8679 (1/24E05-SCRF was received at ATCC Dec.12, 1984. Hybridoma HB8800 (18-9 B10-SCRF 35.3) was received at ATCC onMay 9, 1085 pursuant to the Budapest Treaty.

                  TABLE 3                                                         ______________________________________                                        ATCC Deposits                                                                                           Date of Receipt                                        SCRF of Deposit Myeloma                                                      ATCC No. Ref. No. at ATCC Cell Line                                         ______________________________________                                        HB8800   18-9B10-SCRF35.3                                                                           5/9/88     P3X63-Ag 8.653                                 HB8596 S10F03 8/2/84 SP2-0                                                    HB8595 S22C06 8/2/84 SP2-0                                                    HB8594 P43D09 8/2/84 SP2-0                                                    HB8593 P44E11 8/2/84 SP2-0                                                    HB8679 1/24E05-SCRF 12/12/84   SP2-0                                        ______________________________________                                    

The monoclonal receptors of this invention secreted by hybridomasdesignated S22C06 and S10F03 are particularly preferred monoclonalreceptors. Both preferred monoclonal receptors are IgG1 monoclonalreceptors, having kappa light chains, that immunoreact with theimmunizing polypeptide and with the fes-related oncoprotein having anamino acid residue sequence corresponding to the sequence of theimmunizing polypeptide.

A hybridoma was raised using the ras 23-mer immunogenic, syntheticpolypeptide (ras) shown below:

YREQIKRVKDSDDVPMVLVGNKC.

The monoclonal antibody secreted by that monoclonal antibody binds tothe immunogenic polypeptide and also binds to the 55 K dakton proteinencoded by the ras gene of the Harvey sequence. The monoclonal antibodyrecognizes a 23 K dakton protein in all ras-producing cell lines testedas well as a higher. molecular weight protein. That hybridoma of thisinvention was received at the ATCC on Dec. 12, 1984 and was given thedesignation ATCC HB 8679 (1/24E05-SCRF)).

The hybridomas designated S10F03, S22C06, P43D09, P44E11 and 1/24/E05secrete kappa-light chained, IgG1 monoclonal receptors.

The last-named five hybridomas were prepared from three separate cellfusions. The efficiency of producing hybridomas whose Mabs recognize theimmunogenic polypeptide as well as the corresponding oncoproteinmolecule ligand for the first preparation was 100 percent; i.e., twoMabs (from S10F03 and S22C06) were produced that recognize thepolypeptide, and those two Mabs also recognize the oncoprotein. For thesecond and third preparations, the efficiency, calculated similarly wasabout 20 percent.

Another hybridoma was raised using the erb-B related, 16-mer immunogenicsynthetic polypeptide shown below. The amino acid residue sequence ofthe synthetic polypeptide, from left to right and in the direction fromamino-termius to carboxy-terminus is represented by the formula:

    IMVKCWMIDADSRPKF.

The monoclonal antibody secreted by this hybridoma also binds topolypeptides related to oncoproteins encoded by fes, fms, abl, src andfqr oncogenes.

FIG. 1 illustrates the immunological detection of the p85 oncoproteinligand by the monoclonal receptors secreted by hybridomas S10F03 (ATCCHB 8596) and S22C06 (ATCC HB 8595), using an external standard for thep85 oncoprotein ligand and an influenza hemagglutinin-recognizing Mab asa negative control. FIG. 2 illustrates similar results again using Mabsfrom hybridoma S10F03 as well as Mabs from hybridomas P43D09 (ATCC HB8594), and P44E11 (ATCC HB 8593), and also hybridoma P42C10. Amonoclonal antibody against the Rauscher virus protein denominated gp70[Niman and Elder in Monoclonal Antibodies and T Cell Products, above]was used as a negative control.

FIG. 3 further illustrates the specificity of the monoclonal receptorsof this invention. There, CCL64 mink cells (lanes B and C) or MSTF cellsinfected with FeLV-B and FeSV (lanes A and B) were radioactively labeledwith ³² P. Extracts from the labeled cells were then incubated witheither a goat antiserum against the p15 protein encoded by the gagportion of the v-fes gene and expressed as the protein precursordenominated pr65 (lanes A and B) or with tissue culture supernatant fromhybridoma S10F03 (lanes C and D).

As can be seen, the Mab of this invention from hybridoma S10F03 boundonly to the p85 oncoprotein ligand (lane C), while the goat anti-p15serum bound to both the pr65 and p85 fusion oncoproteins from theinfected cells (lane A). No proteins were bound from the uninfectedcells (lanes B and D). These results and, by analogy, discussion of theassay concerning FIG. 13, confirm that the Mabs of this invention bindonly to the oncoprotein ligand (p85) a portion of whose amino acidresidue sequence corresponds to the sequence of the immunogenicpolypeptide used to prepare the hybridoma secreting each Mab.

In similar results, not shown, Mabs from the above five hybridomas alsobound to the 108 K dakton oncoprotein ligand expressed in cellstransformed by GA-FeSV. The oncoprotein ligand encoded by the GA-FeSVstrain is substantially identical in amino acid residue sequence to theoncoprotein ligand encoded by the ST-FeSV strain in the region of theimmunogenically useful polypeptide. See, Hampe et al., Cell, 30, 777-785(1982).

None of the above five Mabs bound to the oncoprotein encoded by thev-fps gene of the Fujinami strain of avian sarcoma virus. The predictedv-fps oncoprotein also contains extensive homologies to the predictedv-fes oncoprotein and differs in the region corresponding to the above12-mer (polypeptide b) only by the substitution of the first and fourthresidues from the amino-terminus of that 12-mer polypeptide; i.e., theamino-terminal serine (S) of the v-fes-related polypeptide andoncoprotein is replaced by a valine (V) in the v-fps-relatedoncoprotein, and the second proline (P) residue from the amino-terminusis replaced by an alanine (A) residue.

The non-binding of the above Mabs to the v-fps-related oncoproteinprovides a basis for distinguishing among expressed oncoproteins intransformed cells, and for assaying for the presence of thev-fes-related oncoprotein ligand in the presence of the v-fps-relatedoncoprotein. That distinction in binding can also be useful in purifyinga mixture of both proteins by affinity chromatography utilizing an Mabof this invention as a portion of an affinity sorbant, as is discussedhereinafter.

The above non-binding of the monoclonal antibodies of this invention tothe v-fps-related oncoprotein also highlights the improvement inspecificity of the monoclonal receptors over previously obtainedoligoclonal receptors. Thus, Sen et al., Proc. Natl. Acad. Sci. USA, 80,1246-1250 (1983), used polypeptide (b) above conjugated to KLH toprepare rabbit oligoclonal antibodies. Those oligoconal antibodies boundto oncoproteins expressed in cells transformed by ST-FeSV, GA-FeSV andFSV (Fuginami sarcoma virus) that contain the v-fes^(ST) v-fes^(GA) andv-fps oncogenes, respectively. It can therefore be seen that thespecificity obtained from the monoclonal receptors of this invention isgreatly improved over that obtained with oligoclonal receptors even whenboth are raised to the same immunogenic polypeptide.

In a similar manner are prepared hybridomas that secrete monoclonalreceptors that bind to oncoprotein molecule ligands, e.g., PDGF, toimmunogenic polypeptides encoded by the retroviral oncogenes denominatedfes, myb, sis, ras, myc and mos, as well as to immunogenic polypeptideswhose sequences correspond to sequences of oncoproteins encoded byoncogenes denominated fps, src, yes, fgr, fms, erb-B, mht, raf, abl andrel, and also to oncoproteins expressed in cells transformed byretroviruses containing those genes. Specific monoclonal receptors ofthis invention bind to an immunogenic polypeptide encoded by the aboveoncogenes.

Some of those oncogenes are named below in Table 4 and are illustratedadjacent to formulae of the polypeptides encoded by those sequences towhich the preferred monoclonal receptors of this invention bind. Theright-hand column illustrates those instances where oligoclonal antiseraraised to an enumerated polypeptide have been shown to containantibodies (receptors) that bind to the oncoprotein that contains anamino acid residue sequence corresponding to the sequence of thepolypeptide using a Western blot analysis. Binding is shown by a plussign (+), and oligoclonal receptor-containing antisera for which a plussign is listed exhibited a 50 percent binding titer as described before.The designation "NT" indicates that a rigorous binding study has notbeen conducted. The polypeptide formulae contain the amino acid residuesequences shown, illustrated from left to right and in the directionfrom amino-terminus to carboxy-terminus.

                  TABLE 4                                                         ______________________________________                                                                     Binding of                                                                                      Oligoclonal                      Onco-                                                     Antisera to                                     gene    Polypeptide Sequence                                                 Oncoproteins                                     ______________________________________                                        fes   SDVWSFGILLWETFSLGASPYPNLSNQQTR;                                                                      +                                                   -              SPYPNLSNQQTR;                            +                     -               IHRDLAARNCLVTEKN;                        NT                   -              IGRGNFGEVFSG;                            +                     -             LMEQCWAYEPGQRPSF;                        +                      -            VPVKWTAPEALNYGR; and                     +                       -              SSGSDVWSFGILLWE;                         NT                    - myb                   RRKVEQEGYPQESSKAG;                       +                                        -                RHYTDEDPEKEKRIKELEL; and                                                  +                                    -               LGEHHCTPSPPVDHG;                         NT                   - sis         RKIEIVRKKPIFKKATV; and                   +                      -         RVTIRTVRVRRPPKGKHRKC;                   +                           - ras         YREQIKRVKDSDDVPMVLVGNKC;                 +                      -      KLVVVGARGVGK;                            +                             -               KLVVVGASGVGK; +                                               -       KLVVVGAVGVGK; and                        NT                           -      KLVVVGAGGVGK;                            +                             - myc        CDEEENFYQQQQQSEL; +                                              -               PAPSEDIWKKFEL; +                                              -                 LPTPPLSPSRRSGLC; +                                          -               CDPDDETFIKNIIIQDC;                       NT                   -              CSTSSLYLQDLSAAASEC;                      +                     -             CASQDSSAFSPSSDSLLSSTESSP; and            NT                     -            CTSPRSSDTEENVKRRT;                       +                       - mos         LPRELSPSVDSR;                            +                      -       RQASPPHIGGTY; and                        +                            -      TTREVPYSGEPQ;                            +                          B    ENDTLVRKYADANAVCQ;                       +                                  -     LGSGAFGTIYKG (C); and                    NT                             -    IMVKCWMIDADSRPKF;                        +                               - PDGF-2    SLGSLTIAEPAMIAECK;                       +                        -   RKIEIVRKKPIFKKATV; and                   +                                -  RVTIRTVRVRRPPKGKHRKC;                    +                                 - PDGF-1    SIEEAVPAECKTR;                           +                        - a       LMRACWQWNPSDRPSF;                         NT                        - fms                            FMQACWALEPTRRPTF;                                                          NT                                              - src        LMCQCWRKDPEERPTF; NT                                             -             LGQGCFGEVWMG; and                         +                     -            CGSSKSKPKDPSQRRRS;                        NT                     - fgr        AMEQTWRLDPEERPTF.                         NT                  ______________________________________                                    

The polypeptides useful for inducing the production of oligoclonalreceptors, and ultimately for production of monoclonal receptors, arepreferably linked to a carrier molecule, as discussed herein whereinpolypeptides linked to KLH have been utilized throughout as illustrativepolypeptide-carrier conjugates. For polypeptides that contain fewer thanabout 35 amino acid residues, it is preferable to use a carrier for thepurpose of inducing the production of oligoclonal and monoclonalreceptors. Polypeptides containing about 35 to about 40 amino acidresidues may be used alone, without linkage to a carrier, to inducereceptor production, although it is still preferable to utilize acarrier for producing those receptors. Thus, the receptors may beinduced by or raised to a polypeptide alone, or linked to a carrier.

B. Immunization Binding Studies

As noted several times, the polypeptides utilized in raising oligoclonalantibodies and hybridomas that secrete monoclonal antibodies arethemselves immunogenic and antigenic, and those properties providecriteria for identifying useful polypeptides for hybridoma preparation.The discussion below relates to studies with oligoclonal antibody(receptor)-containing antisera induced by or raised to polypeptides usedin the preparation of hybridomas that secrete monoclonal receptors(antibodies) to oncoproteins encoded by the ras, sis, erb-B and myboncogenes. As will be described, the sis-related polypeptide inducesproduction of oligoclonal receptors that bind not only to thepolypeptide, but also to a corresponding oncoprotein, humanplatelet-derived growth factor (PDGF). The oligoclonal antibodies soprepared exhibited the before-described 50 percent binding titer to theimmunizing polypeptide, thereby indicating that monoclonal antibodies(receptors) of this invention may also be prepared by fusion of theantibody-producing splenocytes with cells of a suitable myeloma line.

PDGF isolated from platelets consists of two chains that areapproximately sixty percent homologous at the amino-terminal end. One ofthose chains (PDGF-2) is virtually identical to a portion of the simiansarcoma virus (v-sis) gene product (p28^(sis)). Sequencing of the humanc-sis and v-sis terminate at the same position and the PDGF-2 moleculeoriginates from a larger precursor which has extensive homology withp28^(sis). The homology between p28^(sis) is and PDGF-2 begins at aminoacid residue 67 of p28^(sis) and the amino-terminus of PDGF-2, and hasrecently been extended to the predicted carboxy-terminus of p28^(sis)via the isolation and sequencing of a human c-sis clone. Josephs et al.,Science, 223, 487-491 (1984).

p28^(sis) is rapidly cleaved to generate p20^(sis) which presumably hasthe same amino terminus as PDGF-2. Within the region coding forp20^(sis) and PDGF-2 there are eight amino acid changes that can beplaced into three regions. The two changes near the amino terminus areconservative, five changes are clustered near the center of themolecule, and one change is located in the carboxyl-terminal portion.

Two exemplary polypeptides were prepared. The first, denominatedpolypeptide (c), corresponds in amino acid residue sequence to residues139 through 155 of the predicted sequence of the simian sarcoma virustransforming protein denominated p28^(sis). Devare et al., Proc. Natl.Acad. Sci. USA, 80, 731-735 (1983). The sequence of polypeptide (c) alsocorresponds to the sequence of positions 73 through 89 from theamino-terminus of the protein chain denominated PDGF-2 of humanplatelet-derived growth factor, as noted before. The second, denominatedpolypeptide (d), corresponds in amino acid residue sequence to residues2 through 18 of the predicted sequence of the transforming protein ofthe avian myeloblastosis virus (v-myb) oncoprotein. Rushlow et al.,Science, 216, 1421-1423 (1982). The amino acid residue sequence ofpolypeptides (c) and (d) are shown below, from left to right and in thedirection from amino-terminus to carboxy-terminus:

    ______________________________________                                        polypeptide (c)    RKIEIVRKKPIFKKATV;                                         Polypeptide (d)        RRKVEQEGYPQESSKAG.                                     ______________________________________                                    

Each of the polypeptides was synthesized and bound to KLE using a Cysresidue of their carboxy-terminii (not shown in the above formulas), andeach resulting conjugate was then used to immunize mice as discussedgenerally in the Materials and Methods section. As can be seen from anexamination of FIG. 4, sera raised to polypeptide (c) containedoligoclonal receptors that bind to polypeptide (c) as well as to KLH,and sera raised to polypeptide (d) contained oligoclonal receptors thatbind to polypeptide (d) and to KLH. Neither serum contained receptorsthat cross-react and bind to the polypeptide not used to raise them.

Extracts from outdated human platelets were used to obtain partiallypurified samples of PDGF. As already noted, PDGF is an oncoproteinhaving an apparent molecular weight of about 30 K daktons that can bereductively cleaved into two high molecular weight polypeptides ofsimilar apparent molecular weights, and designated PDGF-1 and -2.

FIG. 5 shows the results of Western blot analysis of PDGF using theoligoclonal receptor-containing antisera raised to polypeptides (c) and(d), as is discussed in more detail in the description of that figure;the antiserum raised to polypeptide (d) being used as a negativecontrol. As can be seen from an examination of FIG. 5; the oligoclonalreceptor-containing serum raised to the sis-related polypeptide,polypeptide (c), bound to three proteinacious moietities (lane 2). Oneof those moieties has an apparent molecular weight of about 30 K daktonsand two of about 16-18 K daktons each. Lane 4 also illustrates bindingby oligoclonal receptors contained in the anti-sis-related polypeptideserum. As expected, only non-specific binding was shown by oligoclonalreceptors raised to the myb-related polypeptide, polypeptide (d), (lanesand 5).

Presuming that the amino acid residue sequence of PDGF-1 and -2 arecolinear with the sequence of p28^(sis), the amino acid residue sequenceof the polypeptide (c) corresponds to positions 67 through 83, and 73through 89 of PDGF-1 and -2, respectively. The amino acid residuesequence of residues 73 through 80 of PDGF 2 has been determined[Doolittle et al., Science, 221, 275-277 (1983)] and all of the thoseresidues are identical to the first (amino-terminal) eight residues ofpolypeptide (c). In addition, a polypeptide from PDGF and correspondingto residues 147 through 155 of the p28^(sis) oncoprotein has beensequenced [Waterfield, Nature, 304, 35-39 (1983)], and of the nineresidues so far identified, all are identical to the correspondingresidues of polypeptide (c). Thus, sixteen of the seventeen residues ofpolypeptide (c) are identical to and in the same sequence as residues inboth PDGF, derived from humans, and p28^(sis) derived from a line ofretrovirus-transformed cells.

The above results thus illustrate the immunogenicity and antigenicity oftwo additional polypeptides useful for immunizations leading to thepreparation of hybridomas that secrete monoclonal receptors of thisinvention. Those results also show that the oligoclonal receptors raisedto polypeptide (c) also bind to an oncoprotein; i.e., PDGF, PDGF-1 andPDGF-2.

Additional synthetic polypeptides representing various regions of bothPDGF sequences were made. The amino-terminii of PDGF-1 and PDGF-2, aswell as the central and carboxy-terminal portion of PDGF-2 weresynthesized, conjugated to the immunogenic carrier keyhole limpethemocyanin (KLH), and injected into mice to induce production of .oligoclonal receptor-containing antisera that exhibited thebefore-described 50 percent binding titer.

The polypeptide representing the unique region of PDGF-2 contains thefirst 17 amino acids of this sequence and will be called PDGF-2(1-17),wherein the parenthesized numerals indicate the amino acid residues ofthe corresponding molecule numbered from amino-terminus. The uniqueregion of PDGF-1 is represented by a polypeptide PDGF-1(1-12), thatcontains the first 12 amino acids of that sequence. Six of those 12amino acids are shared with PDGF-2 but only three are consecutive, asnoted before. The third polypeptide, PDGF-2(73-89) is also referred toherein as polypeptide (c). It represents the predicted amino acidresidues 139-155 of p28^(sis) and contains an additional cysteine at itscarboxy-terminus for coupling purposes. This polypeptide when coupled toKLH induced production of antibodies that recognize the reduced subunitsof purified PDGF, proteins of MW 31,000, 30,000, 21,000 and18,000-14,000 in a platelet extract, and a 56K dalton protein inSSV-infected marmoset cells. The fourth polypeptide, PDGF-2(126-145),was also predicted by the v-sis sequence (residues 192-211 ofp28^(sis)). Amino acid sequences of these polypeptides have beenillustrated hereinbefore.

To analyze the specificity of the oligoclonal receptor-containingantisera generated against these synthetic polypeptide conjugates, PDGFwas probed with these antisera. Purified PDGF was reduced andelectrophoresed into a polyacrylamide gel, and then onto nitrocellulose(FIG. 6, lanes A-F) using a Western blot procedure. In lanes A and B,two antisera directed against PDGF-1(1-12) immunoreacted with a proteinof approximately 18,000 daltons. Sequence analysis of purified PDGFindicates the majority of the PDGF-1 chain migrates at this position(Antonaides, et al., Science, 220, 963-965 (1983)]. The weakness of thereactivity with these antisera suggests the amino-terminal end of PDGF-1may not be readily accessible for antibody binding.

In contrast, antiserum against the amino-terminus of PDGF-2(1-17) (laneC) readily detected a protein migrating at about 18,000 and 14,000daltons, consistent with sequence analysis of PDGF-2 (Antonaides et al.,supra.).

The antisera induced by PDGF-2(73-89) produced the same activities(lanes D,E) as seen in lane C. In contrast, antisera againstPDGF-2(126-145) did not have detectable activity against purified PDGF.

Since the sequence of the PDGF-2(126-145) polypeptide differs fromc-PDGF at position 145 (Josephs, et al., supra), it is possible thatthis amino acid residue change is contained within the epitopic site.This is unlikely because the polypeptide is 20 amino acid residues longand the change is only on the carboxy-terminal position that is used tocouple the polypeptide to the KLH carrier protein. The lack of activityis thus not due to generation of oncopolypeptide-specific antibodiesbecause this antiserum reacts with cell-derived PDGF-like molecules. The14,000 to 18,000 dalton size of the detected PDGF in purifiedpreparations suggests that most of this material is missing thecarboxy-terminal end of the predicted sequence of p28^(sis), which wouldremove all or part of the PDGF antigenic-site recognized by thisantiserum.

In order to determine if PDGF-like proteins might also be synthesized inother transformed cell lines, extracts were made and were immunoreactedwith various oligoclonal receptor-containing antisera againstPDGF-related polypeptides. In FIG. 7, the SSV-transformed NIH 3T3 cellswere probed with an oligoclonal receptor-containing antiserum induced byPDGF-1(1-12) (lanes A-C, F-H and K-M) and by PDGF-2(73-89) (lanesD,E,I,J,N and O). Of the two sera against PDGF-2(73-89) (FIG. 6, lanes Dand E), the serum used in FIG. 6, lane D produced a somewhat weakeractivity with purified PDGF. However, as seen in lane D of FIG. 7, astrong reactivity with a protein of approximately 70,000 daltons wasobserved that was blocked by preincubation with the immunizingpolypeptide, PDGF-2(73-89)(lane E), but was not blocked by preincubationof the antiserum with PDGF-1(1-12).

Thus, the specific reactivity with these oncoproteins by both antiserademonstrates that this is not a fortuitous cross-reactivity with a smallregion of PDGF, but that this molecule contains sequences homologous toat least the amino-terminus of PDGF-1 and the central region of PDGF-2.The amounts of p28^(sis) and p20^(sis) is were below the level ofdetection with this anti-PDGF-2(73-89) serum. Similar results wereobtained with additional antisera, although overexposure didoccasionally show a 20,000 dalton band was specifically detected (datanot shown).

Analysis of extracts of two other unrelated transformed cells with theseantisera gave similar results. The TRD1 cell line is a spontaneouslytransformed Balb/3T3 cell line [Bowen-Pope et al., Proc. Natl. Akcad.Sci. U.S.A., 81, 2396-2400 (1984)]. This line also expresses a 70,000dalton protein as well as a more immunologically related protein ofapproximately 100,000 daltons (FIG. 7, lanes G-I). A third cell line,MSTF, and a mink lung line (CCL64) productively infected with FeLV-B andthe Synder-Theilen strain of FeSV, also expresses the same size proteinFIG. 7, lanes K-O.

In addition to the 70,000 dalton oncoprotein, an oligoclonalreceptor-containing antiserum against PDGF-1(1-12) detected proteins ofapproximately 53,000 daltons (data not shown). These proteins are notserum contaminants because they are detected in extracts of cells thathave been grown for one month in the absence of serum and are found inserum free media conditioned by the TRD1 cell lines. All cell linesstudied contain these two PDGF-like proteins. (See also discussion ofFIG. 11 in "Brief Description of Figures").

The expression of PDGF-like molecules in a broad spectrum of cells,including cells that are not oncogenically transformed (normal diploidrat smooth muscle and human lung fibroblasts), indicates that otherprocesses are involved in transformation. Although all of the cell linescontained 70,000 and 53,000 dalton proteins detected with oligoclonalreceptor-containing antisera induced by PDGF-1(1-12), the cells werequite heterogeneous with regard to size and intensity of other proteinsdetected with antisera directed against determinants predicted by thesequence of the PDGF-2 region (data not shown). The nature of thesedifferences is presently unknown.

In a similar manner, each of the four immunogenic polypeptides,denominated (e-h) below, may be used to induce oligoclonal receptorsthat bind to those immunogenic polypeptides used to induce theirproduction as well as to each of two oncoproteins encoded by the rasoncogene. The sequences of those four ras-related polypeptides, in thedirection from left to right and from amino-terminus tocarboxy-terminus, are represented by the formulas:

    ______________________________________                                        polypeptide e      KLVVVGARGVGK;                                              polypeptide f KLVVVGASGVGK;                                                      - polypeptide g KLVVVGAVGVGK;                                                 - polypeptide h KLVVVGAGGVGK; or                                           ______________________________________                                    

by the combined formula:

    ______________________________________                                                polypeptide (e-h)                                                             KLVVVGAR (S, V, G) GVGK;                                              ______________________________________                                    

wherein the amino acid residues in parentheses are each an alternativeto the immediately preceding amino acid residue in the formula. Theoligoclonal receptors so prepared have a 50 percent binding titerdilution of more than 1:400 after two immunizations, as describedbefore, in about a one month period. Additionally, each ras-relatedoligoclonal receptor induced by polypeptides (e), (f) and (h) have beenshown to bind to an oncoprotein present in lysed cell extracts from (a)human T24 bladder carcinoma cells and also (b) Harvey murine sarcomavirus-infected mouse 3T3 cells (data not shown).

As is seen in FIG. 12, each of the two immunogenic polypeptidesdenominated below (k and l) may be used to induce oligoclonal receptorsthat bind to those immunogenic polypeptides used to induce theirproduction as well as to each of two oncoproteins encoded by thev-fes^(ST) oncogene. The sequence of the two v-fes-related polypeptides,in the direction from left to right and from amino-terminus tocarboxy-terminus are represented by the formulae:

    ______________________________________                                        polypeptide k      LMEQCWAYEPGQRPSF                                           polypeptide l IGRGNFGEVFSG.                                                   ______________________________________                                    

The oligoclonal receptors induced by polypeptides (k) and (l) have beenshown to bind to an oncoprotein present in supernatant from cells ofhuman T24 bladder carcinoma and a spontaneously transformed mouse 3T3cell line (Lanes A and C).

As shown in FIG. 13, a protein related to the ras oncogene was detectedby a monoclonal antibody (from hybridoma ATCC HB 8679) raised to a rassynthetic peptide corresponding to positions 96-118 of v-ras^(Ha). Theprotein is detected in lane A and blocked by preincubation with theimmunizing peptide (lane B). Thus, the preincubation with the immunizingpolypeptide blocked the strongly reactive oncoprotein.

The use of monoclonal receptors of this invention such as those raisedto the sis-(PDGF)related polypeptide (c), or to the fes-relatedpolypeptides (a), (b) (k) or (1), or to the ras-related polypeptides(e-h) or to the other oncoprotein-related polypeptides disclosed hereinin the affinity sorbants described below provides a convenient and lessarduous means for preparing naturally occurring proteinaceous materialsthat are otherwise difficult to obtain in purified form such as PDGF.Thus, rather than having to go through the long procedure to obtainpurified PDGF, discussed hereinafter, one may, for example, merely lysethe cells, centrifuge, pour the supernatant through an affinity sorbantcolumn containing bound anti-polypeptide (c) receptor, and elute thepurified protein after dissociating the formed, reversible ligandcomplex. While some additional proteinaceous material may benon-specifically bound to the affinity sorbant column, the isolation ofpurified proteins that are otherwise difficult to obtain in such form isgreatly enhanced using such sorbants.

The antisera to the conserved sequences described above react withproteins in a wide variety of transformed cell lines. The antiserareadily detected oncogene-related proteins that were five- to fifty-foldmore concentrated in th urine of cancer patients and pregnant women thanin normal controls. Unique patterns of expression were detected invarious malignancies and during different gestational stages ofpregnancy.

Anti-peptide antibodies are particularly suited for detecting proteinsimmunologically related to-sequenced oncogenes [wong et al., Proc. NatlAcad. Sci., USA 78, 7412-7416 (1981)]. Since they are sequence specific,anti-peptide antibodies can be directed toward highly conserved regionsof proteins to maximize the probability of identifying related moleculeswhich may have similar functions. Because immune recognition of proteinsby anti-peptide antibodies need not be highly dependent upon antigenconformation, one can identify proteins that are not detected byanti-protein antibodies, the bulk of which are directed againstdeterminants unique to the folded protein. Finally, the binding ofanti-peptide antibodies is relatively insensitive to alteration orfragmentation of the target antigen such as might occur in bodily fluidsor secretions.

In Table 4, the synthetic peptides used to, generate the antibodies arelisted together with related sequences of other oncogenes. The raspeptide is the H-ras sequence located 37-59 amino acids downstream fromthe threonine residue auto-phosphorylated by p21 encoded by v-H-ras orv-K-ras. The sequence is identical in c-H-ras, c-N-ras, and differs fromc-K-ras by one conserative amino acid change. Capon et al. Nature(London) 304, 507 (1983) The sequence of PDGF-2 used to generate the sismonoclonal antibodies is located at the amino-terminus of the chain andis homologous to the first 12 amino-acids of the other chain (PDGF-1) ofplatelet-derived growth factor. The fes peptide constitutes residues754-769 of the 85,000 dalton fusion protein and is 79-94 amino acidsdownstream from the major tyrosine phosphosylation site. The peptidesused for this study were selected because they represent highlyconserved regions of the respective oncogene families.

The antisera to these conserved sequences react with proteins in a widevariety of transformed cell lines. The reactivity of the three antiserawith proteins of a mink lung line transformed by feline sarcoma virusare shown in FIG. 1. Antibodies against the sis-peptide detect a 20,000dalton protein in SSV-transformed NRK cells as well as a sis-relatedprotein of approximately 56,000 dlatons in the mink lung line (lane 1).Antibodies against the ras peptide detect a major protein ofapproximately 21,000 daltons and a minor protein of approximately 30,000daltons in the cell extract (lane 2). The antiserum against the fesprotein detects the 85,000 dalton gag-fes fusion protein as well as a40,000 dalton protein (lane 3).

In FIG. 15, the reactivity of these antisera with urinary proteins froma variety of patients is demonstrated. The sis antisera detects proteinsof 56,000, 31,000 and 25,000 daltons in urine concentrates (Panel A).

The antibody binding to all three proteins is blocked by priorincubation with the sis peptide (Panel B) but not by incubation with theras peptide (Panel A). The concentrations of the detected proteins arefive to fifty fold higher than normal individuals (see below). Allurines studied contained the three sis-related proteins except for thesample from the patient with lymphoma which is missing the 56,000 daltonprotein (lane 4).

The somewhat faster mobilities of p56^(sis) (Panel A, lanes 1 and 2) inthe urine from the donors with multiple myeloma and gastric cancer isdue to excess albumin in these samples while the distortion of the lowermolecular weight proteins in lane 1 are due to excessive amounts ofantibody light chain.

In Panel C the various ras-related proteins detected in urine samplesare displayed. Proteins are approximately 100,000 and 55,000 dalton aredetected (Panel C, lane 2-4). Again, the specificity of the antiserumwas demonstrated by blocking the activity by preincubation with the raspeptide (Panel D) but not by preincubation with the sis peptide (PanelC).

The 55,000 dalton ras-related protein is different from the 56,000dalton sis-related protein (see below) and displays different reactivitypatterns in each sample. The protein is not detectable in Panel C, lane1 (gastric cancer) while four bands of almost equal intensity are seenin lane 2 (38 weeks pregnant).

A strongly reactive doublet is visualized in lane 3 when urine from apatient (donor) with breast cancer was probed. A minor band atapproximately 35,000 daltons is associated with high concentrations ofthe 55,000 dalton protein. In lane 4, a single 55,000 dalton band wasdetected.

Proteins of approximately 21,000 daltons were detected in all 4 lanes ofPanel C. These smaller proteins were present at similar concentrationsalthough the mobility of the protein in Panel C, lane 1 is slightlyslower. This altered mobility may be significant because of the effectof changes at amino acid residue position 12 on the electrophoreticmobility of ras encoded proteins. The binding detected at 25,000 daltonsis difficult to interpret due to comigration with antibody light chain.

In Panel E, the 35,000 and 40,000 dalton fes-related proteins are shown.The binding was blocked by preincubation with the immunizing fes peptide(Panel E, lane 1) but not incubation with the ras peptide or peptidesrepresenting the homologous sequences in erb B or abl proteins (Panel E,lanes 2-4).

In summary, the 3 antisera described above specifically detect 8different proteins in urine, 3 sis-related proteins (p56^(sis),p31^(sis), and p25^(sis)) 3 ras-related proteins (p100^(ras), p55^(ras),and p21^(ras)) and 2 fes-related proteins (p40^(fes), p35^(fes))

In FIG. 23, the frequencies of detection of oncogene-related proteins inthe 51 control (normal; free from diagnosed neoplastic disease) urinesamples or 189 samples from patients (donors) with a variety ofmalignancies are listed. Similar, frequencies in 260 urine samples frompregnant women are shown in FIG. 24. The amount of oncogene-relatedproteins in the urine was estimated using immunoblots, and was placedinto 1 of four categories: undetectable, detectable, 5-15-fold elevated,and greater than 15-fold elevated.

The types of malignancies in which more than 10 samples were tested arelisted individually. The remaining types are listed as a composite.

p21^(ras) was detected in approximately 70% of all tumor samples.However, similar frequencies were found in apparently normalindividuals. In contrast to the elevated levels of the ras- andfes-related proteins found in urine of breast cancer patients, bladderand prostate cancer patients frequently secrete elevated levels of the56,000 dalton sis-related protein. This protein was detected in theabsence of the ras- and fes-related proteins described above (FIG. 15,lanes 1, 2, Panels A-C). In addition to the 56,000 dalton sis-relatedprotein, these patients frequently had elevated levels of the 31,000and/or 25,000 sis-related proteins. In further contrast, urine from apateint with a benign prostate nodule did not contain elevated levels ofthese oncogene-related proteins (FIG. 17, lane 3, Panels A-C).

High levels of the smaller proteins were also found frequently in urinefrom patients with lung and cervical cancer as well as non-Hodgkinslymphomas (see FIG. 23). In these latter patients, the elevated 31,000and/or 25,000 sis-related proteins were found in the absence of the56,000 dalton protein (FIG. 13, lane 4, Panel A-C).

Thus, in the urine samples from cancer patients three unusual patternshave been observed. A subset of the breast cancer patients have elevatedlevels of p55^(ras) in conjunction with p40^(fes) and/or p35^(fes).Patients with bladder and prostate cancer excrete increased amounts ofall three sis-related proteins in the absence of p55^(ras), p40^(fes),and p35^(fes). Finally, a subset of lung cancer and lymphoma patientsexcreted elevated levels of only the lower molecular weight sizes thesis-related proteins. As can be seen from FIGS. 15-18 as well as FIG.23, patterns of expression correlate with disease states better thanexcretion of high levels of a single oncogene-related protein. Inapparently normal individuals, elevated levels of these proteins arerarely detected.

The proteins described herein are immunologically related to oncogeneproteins based upon the highly specific reactivity of the variousanti-peptide antisera. However, of the eight proteins described, onlytwo (p21^(ras) and p31^(sis)) represent oncogene-encoded whole proteins.

The p21^(ras) protein has GTP binding activity. Thus, p21^(ras) isintimately involved with cell division and therefore it is notsurprising that the protein is readily detected in most urine samples.

Similarly, elevated levels of transcripts specific for H-ras or K-rashave been detected in a wide variety of malignancies as is shown herein.Furthermore, antisera to ras-related products have also detectedelevated expression in tumor tissues. Here, the most striking elevationof this protein was found in the urine of malignancies.

p31^(sis) protein which is one of the chains of the platelet-derivedgrowth factor (PDGF) was also detected. Although PDGF-1 chain is only18,000 daltons when isolated from platelets, comparison of the humanc-sis sequence with v-sis indicates the 18,000 dalton protein originatesfrom a larger precursor protein. Indeed, analysis of a partiallypurified platelet extract reveals a protein of approximately 31,000dalton. Since PDGF has potent mitogenic activity and is released fromplatelets at the site of tissue injury, one of the physiologicalfunctions of PDGF is thought to be wound healing. In addition, PDGF-likematerial is secreted from a number of transformed cell lines andsecretion appears to be developmentally regulated in smooth musclecells. Thus, p31^(sis) like p21^(ras) may be physiologically important,and it is not surprising that it is present in the urine in normal andabnormal states.

In addition to the oncogene encoded proteins of expected molecular size,additional proteins were detected in this study. It is not likely thattheir presence is due to spurious cross-reactivities since they areuniquely present in certain cancers as well as during pregnancy.Further, the reaction of the antibodies with these proteins wasinhibited specifically with the appropriate synthetic immunogens. Sincethe peptides used as immunogens represent conserved sequences amongoncogene families, these additional proteins may represent members ofthese gene families. The expression of these genes may come undercoordinate control during neoplasia or pregnancy. Regardless of theorigin of these proteins, the fact that they are uniquely expressedduring neoplasia and pregnancy makes them important markers.

III. DIAGNOSTIC SYSTEMS AND METHODS

A diagnostic system, preferably in kit form, comprises.yet anotherembodiment of this invention. This system is useful for assaying for thepresence of an oncoprotein ligand by the formation of an immunereaction. This system includes at least one package that containsbiologically active monoclonal receptor molecules of this invention.Thus, the receptor binds to (a) a polypeptide containing about 7 toabout 40, and preferably about 10 to about 30, amino acid residues in anamino acid residue sequence that corresponds to a portion of the aminoacid residue sequence of an oncoprotein ligand encoded by a gene of aretrovirus, and (b) the oncoprotein ligand encoded by a retroviral gene.

When a predetermined amount of moncolonal receptor molecules is admixedwith a predetermined amount of an aqueous composition containing anoncoprotein ligand, an immunological reaction occurs that forms acomplex between the receptor and the ligand (antibody and antigen).Exemplary aqueous compositions containing an oncoprotein include,without limitation, cell lysates, serum, plasma, urine and amnioticfluid.

In addition, it is particularly valuable to utilize a screening withantisera to more than one oncogene-related translation product. Thus,assay methods set forth herein can be performed on a group of body fluidsample aliquots taken from a single donor to yield accurate informationregarding a neoplastic state, gestational stage or the like.

Admixture between receptor and ligand occurs in an aqueous composition.However, either the receptor or ligand can be substantially dry andwater-free prior to that admixture. Thus, a solution of the receptor inhybridoma supernatant, ascites fluid or buffer can be admixed with anaqueous cell extract to admix the reagents from two aqeuouscompositions; the receptor can be coated on the walls of a microtiterplate and then admixed with a cell extract or serum containing theligand; or the ligand can be coated on microtiter plate walls, on anitrocellulose sheet after transfer from an acrylamide gel or the like,or can be present in a tissue section, and hybridoma supernatant,ascites fluid or a buffer solution containing the receptor admixedtherewith.

The use of exemplary diagnostic systems and methods of this invention isillustrated in the discriptions of the Figures. There, oncoproteinligands coated onto nitrocellulose and then admixed with a receptor ofthis invention are discussed in relation to FIGS. 1, 2, 10 and 13, whilea cell extract incubated with hybridoma supernatant to form animmunological complex is discussed regarding FIGS. 3 and 13.Oncoproteins from urine samples are discussed in FIG. 9, 10 and 14-19.

Receptors are utilized along with an "indicating group" or a "label".The indicating group or label is utilized in conjunction with thereceptor as a means for determining whether an immune reaction has takenplace and an immunological complex has formed, and in some instances fordetermining the extent of such a reaction.

The indicating group may be a single atom as in the case of radioactiveelements such as iodine 125 or 131, hydrogen 3, sulfur 35, carbon 14, orNMR-active elements such as fluorine 19 or nitrogen 15. The indicatinggroup may also be a molecule such as a fluorescent dye like fluoresein,rhodamine B, or an enzyme, like horseradish peroxidase (HRP) or glucoseoxidase, or the like.

The indicating group may be bonded to the receptor as where an antibodyis labeled with ¹²⁵ I. The indicating group may also constitute all or aportion of a separate molecule or atom that reacts with the receptormolecule such as HRP-linked to rabbit anti-mouse antibodies where theantibody receptor was raised in a mouse, or where a radioactive elementsuch as ¹²⁵ I is bonded to protein A obtained from Staphylococcusaureus.

Where the principal indicating group is an enzyme such as HRP or glucoseoxidase, additional reagents are required to visualize the fact that animmune reaction has occurred and the receptor-ligand complex has formed.Such additional reagents for HRP include hydrogen peroxide and anoxidation dye precursor such as diaminobenzidine. Additional reagentsuseful with glucose oxidase include ABTS dye, glucose and HRP.

The terms "indicating group" or "label" are used herein to includesingle atoms and molecules that are linked to the receptor or usedseparately, and whether those atoms or molecules are used alone or inconjunction with additional reagents. Such indicating groups or labelsare themselves well-known in immunochemistry and constitute a part ofthis invention only insofar as they are utilized with otherwise novelreceptors, methods and/or systems.

An indicating group or label is preferably supplied along with thereceptor and may be packaged therewith or packaged separately.Additional reagents such as hydrogen peroxide and diaminobenzidiene mayalso be included in the system when an indicating group such as HRP isutilized. Such materials are readily available in commerce, as are manyindicating groups, and need not be supplied along with the diagnosticsystem. In addition, some reagents such as hydrogen peroxide decomposeon standing, or are otherwise short-lived like some radioactiveelements, and are better supplied by the end-user.

The diagnostic system may also include a solid matrix that may be 96well microtiter plates sold under the designation Immulon II (Dynatech,Alexandria, Va.). The microtiter strip or plate is made of a clearplastic material, preferably polyvinyl chloride or polystyrene.Alternative solid matrices for use in the diagnostic system and methodof this invention include polystyrene beads, about 1 micron to about 5millimeters in diameter, available from Abbott Laboratories, NorthChicago, Ill.; polystyrene tubes, sticks or paddles of any convenientsize; and polystyrene latex whose polystrene particles are of a size ofabout 1 micron and can be centrifugally separated from the latex.

The solid matrix may also be made of a variety of materials such ascross-linked dextran, e.g., Sephadex G-25, -50, -100, -200, and the likeavailable from Pharmacia Fine Chemicals of Piscataway, N.J., agarose andcross-linked agarose, e.g., Sepharose-6B, CL-6B, 4B, CL46 and the like,also available from Pharmacia Fine Chemicals.

The diagnostic system may further include a standard against which tocompare the assay results and various buffers in dry or liquid form for,inter alia, washing microtiter plate walls, diluting the sample,diluting the labeled reagent, or the like.

An assay method for the presence of an oncoprotein ligand in a bodysample from a warm-blooded animal constitutes another aspect of thepresent invention. In accordance with the general assay method, amonoclonal receptor of this invention is admixed in an aqueouscomposition that contains the sample to be assayed for the presence ofan oncopro- tein ligand. Preferably, the monoclonal receptor and bodysample are utilized in predetermined amounts. The admixture so preparedis maintained for a period of time sufficient for an immunoreaction tooccur between the receptor and ligand and an immunocomplex (reactionproduct or immunoreactant) to form. The presence of an immunocomplex isthen determined, and its presence indicates the presence of theoncoprotein ligand in the assayed sample. The presence of animmunocomplex is determined using the before-described labels or byother means well known in immunochemistry for determining the presenceof antibody-antigen complexes.

Specific assay methods are also contemplated. Each of those specificmethods utilizes the above three steps, but the specifics of those assaymethods differ slightly from one another.

Solid phase assays wherein the sample to be assayed is affixed to asolid phase matrix such as a microtiterplate test well or anitrocellulose sheet to form a solid support are particularly preferred.In such instances, admixture of the sample to be assayed and themonoclonal receptor forms a solid-liquid phase admixture. The solid andliquid phases are separated after the before-described maintenanceperiod, and the presence of a ligand-receptor complex is determined bythe presence of receptor bound to the solid support. The relative amountof bound receptor can be determined in many assays, thereby alsoproviding a determination of the amount of oncoprotein ligand that waspresent in the sample assayed.

A receptor molecule of this invention can also be affixed to the solidmatrix to form a solid support. In that instance, the sample to beassayed is admixed to form a solid-liquid phase admixture, the admixtureis maintained as described before, and the presence of an immunocomplexand oncoprotein in the assayed sample are determined by admixture of apredetermined amount of a labeled ligand such as a polypeptide oroncoprotein that is bound by the affixed receptor molecule. Thus, thepresence of a complex formed between the receptor and oncoprotein of thesample provides an amount of labeled ligand binding that is less than aknown, control amount that is exhibited when the sample is free ofoncoprotein being assayed. The relative amount of oncoprotein in thesample can be determined by using an excess of the receptor andmeasuring the lessened binding of the labeled ligand.

A polypeptide or oncoprotein ligand bound by a receptor molecule of thisinvention can also be affixed to a solid matrix to form the solidsupport antigen. A known, excess amount of receptor molecules of thisinvention is admixed with the sample to be assayed to form a liquidadmixture. The liquid admixture so formed is maintained for a period oftime sufficient to form an immunocomplex reaction product, and isthereafter admixed with the solid support to form a solid-liquid phaseadmixture. That admixture is maintained for a period sufficient for theexcess, unreacted receptor molecules present to immunoreact and form acomplex with the solid phase support antigen. The amount of that complexthat is formed is determined, after separation of the solid and liquidphases, using a previously described technique. This method can providea determination as to the presence of oncoprotein in the sample, andalso as to its relative amount, where predetermined amounts of receptorand solid phase ligand are used.

IV. DIFFERENTIAL ASSAY

Liquid body samples can be screened with antisera to more than oneoncogene-encoded protein. The screening can be systematicallyaccomplished in accordance with the assay methods of this invention.

The screening of samples with more than one antiserum provides a patternof oncoproteins present in the sample assayed.

In breast cancer patients, p55^(ras) and p40^(fes) are found to beelevated (FIG. 16) in contrast to the p56^(sis) found in bladder andprostate cancer patients (FIGS. 17 and 18). Also, bladder and prostatecancer patients often demonstrated elevated levels of the 31 K dakton or25K dalton sis-related proteins. In contrast, a donor with a benignprostate nodule did not demonstrate these elevated levels of protein.

High levels of the smaller proteins were also found in patients withlung and cervical cancer as well as non-Hodgkins lymphomas (See FIG.24). In these patients, the elvated 31 K dakton and/or 25K daltonsis-related proteins were found in the absence of the 56 K daktonprotein (See FIG. 18, lane 4, Panels A-C).

Thus, in the urine samples from cancer patients three unusual patternshave been observed. A subset of the breast cancer patients have elevatedlevels of p55^(ras) in conjunction with p40^(fes) and/or p35^(fes). Incontrast, patients with bladder and prostate cancer excrete increasedamounts of all three sis-related proteins in the absence of p55^(ras),p40^(fes), and p35^(fes). Finally, a subset of lung cancer and lymphomapatients excrete elevated levels of only the lower molecular weightsizes of the sis-related proteins. As can be seen from the Figures,patterns of expression correlate with diseased states better thanexcretion of high levels of a single oncogene-related protein. Inapparently normal individuals, elevated levels of these proteins arerarely detected.

The finding of oncogene-related proteins in urine was unexpected and hasnot been previously reported by others. This finding provides a basisfor still another method aspect of the present invention.

In accordance with this method, a sample of urine or a urine concentrateis admixed in an aqueous composition, as described before, with areceptor that immunoreacts with an oncoprotein. The admixture ismaintained for a period of time sufficient for an immunocomplex to form,and the presence of an immunocomplex is determined as described beforein relation to the general assay method and the before-describedspecific methods.

In this method, any receptor known to immunoreact with an oncoproteincan be used. Thus, the receptor molecules can be of polyclonal,oligoclonal or monoclonal origin, and can have been raised to a whole orfusion oncoprotein, or a polypeptide as described herein.

Blotting techniques such as those of the Western blots of the Figuresand so-called "slot blots" wherein the sample is affixed to anitrocellulose matrix as a solid support and where the receptormolecules in a liquid aqueous composition are admixed on thenitrocellulose sheet are preferred techniques for analysis. However,other techniques such as solid phase ELISA and radioimmunoassay (RIA)that utilize microtiter plate wells as solid matrices, and dip stickmethods are also useful.

V. AFFINITY SORBANTS

Affinity sorbants in which the monoclonal receptor molecules of thisinvention constitute the active, binding portions constitute yet anotherembodiment of this invention.

In this embodiment, the monoclonal receptor molecules of this inventionare linked to a solid support that is chemically inert to theoncoprotein ligands to be purified by those sorbants. The phrase"chemically inert" is used herein to mean that a chemical reactionbetween the solid support and the oncoprotein ligands does not occur.However, physical interactions between the solid support and theoncoprotein ligands such as non-specific binding can and do occurbetween them, although such interactions are preferably minimized.

The solid support may be made of a variety of materials such ascross-linked dextran, e.g., Sephadex G-25, -50, -100, -200 and the likeavailable from Pharmacia Fine Chemicals of Piscataway, N.J., agarose andcross-linked agarose, e.g., Sepharose 6B, CL6B, 4B, CL4B and the likealso available from Pharmacia Fine Chemicals or Bio-Gel A-0.5M, A-1.5M,A-50M and the like available from Bio-Rad Laboratories, Richmond,Calif., or polyacrylamide beads, e.g., Bio-Gel P-2, P-30, P-100, P-300and the like also available from Bio-Rad Laboratories. Polyacrylamidebeads have the lowest tendency for non-specific binding among the abovesupports, but also typically have a low porosity that limits theirbinding capacity. The agarose and cross-linked agarose materials arepreferred herein and will be used illustratively as a solid support.

The agarose support is typically activated for linking using cyanogenbromide. The activated support is then washed and linked to the receptormolecules without drying of the activated support. The support-linkedreceptor is then washed and is ready for use. Unreacted reactive groupson the support can be reacted with an amine such as ethanolamine orTris, if desired, although those reactive groups decay quickly.

The affinity sorbant may be used in its loose state, as in a beaker orflask, or it may be confined in a column. Prior to use, it is preferablethat the affinity sorbant be washed in the buffer or other aqueousmedium utilized for oncoprotein purification to eliminatenon-specifically bound proteins or those receptors that were unstablylinked to the support.

An aqueous composition containing an oncoprotein ligand having an aminoacid residue sequence corresponding to the amino acid residue sequenceof the polypeptide to which the linked receptor of the affinity sorbantbinds such as serum or a cell extract is provided, and then admixed withthe affinity sorbant. That admixture forms a reversible, linkedreceptor-ligand complex between the linked receptor and the oncoproteinligand.

The Ligand receptor-ligand complex is then separated from the remainderof the un-complexed aqueous composition to thereby obtain theoncoprotein in purified form linked to the affinity sorbant. When theadmixture takes place in a beaker or flask, this separation can be madeby filtration and washing. When the sorbant is in a column, theseparation may take place by elution of the un-complexed aqueous medium,again, preferably, followed by a washing step.

When the purified protein is desired free from the affinity sorbant, itcan typically be obtained by a variety of procedures. In any of thoseprocedures, the reversible linked receptor-ligand complex is dissociatedinto its component parts of support-linked receptor and oncoproteinligand, followed by separating that ligand from the linked-receptor toprovide the purified oncoprotein free from the affinity sorbant.

The dissociation of the reversible complex may be effected in a numberof ways. A 0.2 molar glycine hydrochloride solution at a pH value ofabout 2.5 is typically utilized. Alternatively, the bound ligand can becompeted away from the linked receptor by admixture of the reversiblecomplex with an excess of the immunogenic polypeptide utilized to raisethe receptor. Such a competition avoids possible denaturation of theligand. Separation of the dissociated oncoprotein ligand from theaffinity sorbant may be obtained as above.

The preparation of affinity sorbants and their use is broadly old.However, such materials and uses that incorporate the receptor moleculesof this invention have not been heretofore available. A detaileddescription of affinity sorbants, their methods of preparation and usewherein the antigen is linked to the support may be found in Antibody asa Tool, Marchalonis and Warr eds., John Wiley & Sons, New York, pages64-67 and 76-96 (1982).

V. MATERIALS AND METHODS

A. Growing of Viruses and Cell Lines

An uninfected mink lung cell line (CCL64), the same line productivelytransformed with the Snyder-Theilen strain of feline sarcoma virus(ST-FeSV) and feline leukemia virus B (FeLV-B) and designated MSTF, aswell as the same line non-productively infected with Gardner-Arnsteinfeline sarcoma virus (GA-FeSV) and designated 64F3C17 were cultured asdescribed in Sen et al., Proc. Natl. Acad. Sci. USA, 80, 1246-1250(1983). A non-producing avian myeloblast cell line, non-productivelyinfected with avian myeloblastosis virus was cultured as described inDuesberg et al., Proc. Natl. Acad. Sci. USA, 77, 5120-5124 (1980). Thenon-producing marmoset cell line, non-productively infected with simiansarcoma virus (SSV) and designated NPV/SiSV and NPVI/SiSV were culturedas described in Devare et al., Proc. Natl. Acad. Sci. USA, 80, 731-735(1983). The avian fibroblast non-productively transformed cell lineinfected with Fujinami sarcoma virus (FSV) was a gift from B. Sefton ofthe Salk Institute, La Jolla, Calif. Uninfected mouse NIH 3T3 fibroblastcells and mouse NIH 3T3 fibroblast cells productively infected withHarvey murine sarcoma virus were cultured as described in Todaro et al.,J. Cell Biol., 17, 299-313 (1963); and Harvey, Nature, 204, 1104-1105(1964). Human T24 bladder carcinoma cells were cultured as described inBubenik et al., Int. J. Cancer, 11, 765-773 (1973).

B. Synthesis of Peptides

Polypeptides were synthesized using solid phase methods as described inMarglin and Merrifield, A. Rev. Biochem., 39, 841-866 (1970), and wereconfirmed by amino acid analyses. Sequence information is derived fromeither amino acid sequencing of the viral protein or predictions basedupon nucleotide sequencing. The sources of the sequence information wereas listed in the footnotes relating to those sequences and theironcogenes.

For polypeptides having fewer than 35 residues that were used inimmunizing inocula, a cysteine residue was added to the amino-terminusor to the carboxyl-terminus of each polypeptide whose correspondingoncoprotein sequence did not contain such a residue. The Cys residueswere used to assist in coupling to a protein carrier as described below.

In preparing a useful synthetic polypeptide by the above solid phasemethod, the amino acid residues were linked to a cross-linked resin(solid phase) through an ester linkage from the carboxy-terminalresidue. When the polypeptide was linked to a carrier via a Cys residue,that Cys residue was conveniently used as the carboxy-terminal residuethat was ester-bonded to the resin.

The alpha-amino group of each added amino acid was typically protectedby a tertiary-butoxycarbonyl (t-BOC) group prior to the amino acid beingadded into the growing polypeptide chain. The t-BOC group was thenremoved by standard techniques prior to addition of the next amino acidto the growing polypeptide chain.

Reactive amino acid side chains were also protected during synthesis ofthe polypeptides. Usual side-chain protecting groups were used for theremaining amino acid residues as follows: O-(p-bromobenzyloxycarbonyl)for tyrosine; O-benzyl for threonine, serine, aspartic acid and glutamicacid; S-methoxybenzyl for cysteine, dinitrophenyl for histidine;2-chlorobenzoxycarbonyl for lysine and tosyl for arginine.

Protected amino acids were recrystallized from appropriate solvents togive single spots by thin layer chromatography. Couplings were typicallycarried out using a ten-fold molar excess of both protected amino acidand dicyclohexyl carbodiimide over the number of millieguivalents ofinitial N-terminal amino acid. A two molar excess of both reagents mayalso be used. For asparagine, an equal molar amount ofN-hydroxy-benzotriazole was added to the protected amino acid anddimethyl formamide was used as the solvent. All coupling reactions weremore than 99% complete by the picric acid test of Gisin, Anal. Chem.Acta. 58:248-249 (1972).

After preparation of a desired polypeptide, a portion of the resulting,protected polypeptide (about 1 gram) was treated with two milliliters ofanisole, and anhydrous hydrogen flouride, about 20 milliliters, wascondensed into the reaction vessel at dry ice temperature. The resultingmixture was stirred at about 4 degrees C. for about one hour to cleavethe protecting groups and to remove the polypeptide Erom the resin.After evaporating the hydrogen flouride at a temperature of 4 degrees C.with a stream of N₂, the residue was extracted with anhydrous diethylether three times to remove the anisole, and the residue was dried invacuo.

The vacuum dried material was extracted with 5% aqueous acetic acid (3times 50 milliliters) to separate the free polypeptide from the resin.The extract-containing solution was lyophilized to provide anunoxidized, synthetic polypeptide.

C. Coupling of Synthetic Polypeptides to Carrier Protein

The unoxidized synthetic polypeptides were coupled to the carrierprotein keyhole limpet hemocyanin (KLH) through a cysteine residue (Cys;C). of the polypeptide with m-maleimidobenzoyl-N-hydroxysuccinimideester as the coupling reagent as described in Green et al., Cell, 28,477 and 487 (1982). Where a Cys residue was a terminal residue in asequence, an additional cysteine residue was not added.

Briefly, as a generalized procedure for each polypeptide, 4 milligramsof KLH in 0.25 millileters of 10 millimolar sodium phosphate buffer (pH7.2) were reacted with 0.7 milligrams of MBS that was dissolved indimethyl fermamide (DMF), and the resulting admixture was stirred for 30minutes at room temperature. The MBS solution was added dropwise toensure that the local concentration of DMF was not too high, as KLH isinsoluble at DMF concentrations of about 30% or higher. The reactionproduct, KLH-MB, was passed through a chromatography column preparedwith Sephadex G-25 (Pharmacia Fine Chemicals, Piscataway, N.J.)equilibrated with 50 millimolar sodium phosphate buffer (pH 6.0) toremove free MBS. KLH recovery from peak fractions of the column eluate,monitored at 280 nanometers, was estimated to be approximately 80%.

The KLH-MB so prepared was then reacted with 5 milligrams of polypeptidedissolved in 1 milliliter of buffer. The pH value of the resultingreaction composition was adjusted to 7-7.5, and the reaction compositionwas stirred at room temperature for 3 hours.

D. Immunization and Fusion

1. Fes-Related Polypeptides

Polypeptides such as those corresponding in amino acid residue sequenceto a portion of the ST-FeSV v-fes oncoprotein were coupled to KLH, andwere used to immunize 129 GIX⁺ mice as described before and in Niman etal., in Monoclonal Antibodies and T Cell Products, Katz ed., (BocaRaton, Fla., CRC Press, Inc., 1982), pp. 21-51. Spleen cells from thoseimmunized mice were fused with SP2/0-Ag14 myeloma cells usingpolyethylene glycol (PEG) 1500 (J. T. Baker Chemco, Phillsburg, N.J.);PEG solutions for fusion were prepared at least one month prior to useto promote fusion efficiency. SP2/0-Ag14 Cells do not produce their ownIg molecules, thereby facilitating isotype analysis and subsequentpurification, such cells also do not produce retroviruses. The fusedcells were then resuspended in 400 milliliters of Dulbecco'shigh-glucose minimal essential medium (Flow Laboratories, Inc.Inglewood, Calif.) containing 10 percent fetal calf serum, 1.0×10⁻⁶molar hypoxanthine, 1×10⁻⁶ molar methotrextate, and 1.6×10⁻⁵ molarthymidine. Next, the cells were plated into 30 microliter plates andgrown as described in Niman et al., Proc. Natl. Acad. Sci. U.S.A., 1982supra.

2. Sis- and Myb-Related Polypeptides

Polypeptides (c) and (d) whose amino acid residues correspond topositions 139-155 of the predicted sequence of simian sarcoma virustransforming protein p28^(sis) and to residues 2-18 of the predictedsequence of the avian myeloblastosis virus oncoprotein were synthesizedand coupled to a KLH carrier as described above. The conjugates soprepared were administered at approximately 50 micrograms of polypeptideper 129 GIX⁺ mouse per injection.

On day 0 (zero), each conjugate was mixed with complete Freund'sadjuvant and injected intraperitoneally. On day 19, each conjugate wasadmixed with alum to provide a concentration of 5 milligrams permilliliter of alum, and injected intraperitioneally. A booster injectionof polypeptide (c) in phosphate-buffered saline was administeredintraveneously on day 62. Serum containing oligoclonal antibodies wastaken by orbital puncture on day 67. After a second alum-containingimmunization of polypeptide (d) on day 41, the booster of polypeptide(d) was similarly administered on day 143 to similarly provideoligoclonal antibodies on day 148. The serum so obtained was tested forthe antigenicity of its receptors as discussed in FIG. 4.

In a similar manner, polypeptides such as those corresponding to thebelow listed amino acid residue sequences were synthesized.

    ______________________________________                                        abl             LMRACWQWNPSDRPSF                                                 - fms       FMQACWALEPTRRPTF                                                  - src       LMCQCWRKDPEERPTF                                                  -        LGQGCFGEVWMG                                                         -         CGSSKSKPKDPSQRRRS                                                   - fgr       AMEQTWRLDPEERPTF                                               ______________________________________                                    

Immunization was carried out in a manner similar to that described forthe sis and myb amino acid residue sequences.

3. Ras- and Erb B-Related Polypeptides

Polypeptides such as those corresponding in amino acid residue sequenceto residues 96-118 of the ras polypeptide from the predicted sequence ofthe ras oncogene of Kirsten murine sarcoma virus and residues the erb Bpolypeptide from the avian erythrablastoma virus were synthesized andcoupled to a KLH carrier as described above. The conjugates so preparedwere administered at approximately 50 micrograms of polypeptide per 129GIX⁺ mouse per injection.

On day 0 (zero), each conjugate was mixed with complete Freunds adjuvantand injected intravenously. On day 5, serum containing oligoclonalantibodies was taken by orbital puncture. The serum so obtained wastested for the antigencity of its receptors as discussed in FIG. 4.

E. Antibody Binding Assay

Hybridomas producing anti-polypeptide antibodies were detected with anenzyme-linked immunoabsorbent assay (ELISA) method as discussed in thedescription of FIG. 4, herein, and in Niman et al., MonoclonalAntibodies and T Cell Products, supra. Briefly, approximately 50micromoles of polypeptide were dried onto microliter plates, fixed withmethanol, and incubated with hybridoma tissue culture supernatant. Afterthorough washing, hydridoma antibody binding was detected using rabbitanti-mouse kappa chain antibody (Litton Bionetics Inc., Kensington, Md.)followed by a glucose oxidase conjugated goat anti-rabbit antisera.Binding was visualized with2,2'-azino-di[3-ethyl-benzothiazoline-sulfonate (6)] (ABTS) dye(Bohringer-Mannheim, Indianapolis, Ind.) in the presence of glucose andhorseradish peroxidase as described in Niman et al., MonoclonalAntibodies and T Cell Products, supra. Isotype was determined bysubstituting various rabbit anti-mouse lambda or heavy chain sera forthe anti-mouse kappa chain as described above.

F. Electrophoretic Transfer and Immunological Detection of Proteins onNitrocellulose

Cell extracts were subjected to polyacrylamide gel electrophoresis, andthe protein was transferred to nitrocellulose (Schleicher and Schuell,Inc., Keene, N.H.) as discussed in the description of FIG. 5, herein,and in Niman et al., Virology, 123, 187-205 (1982). Peroxidase-labeledrabbit anti-mouse IgG serum (Tago, Inc., Burlingame, Calif.) diluted1/1000 was incubated with the transfers for 1 hour at 25 degrees C.followed by washing as described in Niman and Elder, in MonoclonalAntibodies and T Cell Products, above. The bound antibody was visualizedby incubation in 10 millimolar Tris(2-amino-2-(hydroxymethyl)-1,3-propanediol), pH 7.4, 0.009 percent H₂ O₂0.0025 percent 3,3'-dimethoxybenzidine dihydrochloride (Eastman-Kodak,Co., Rochester, N.Y.).

G. Preparation of Purified PDGF

Sixteen units of outdated platelets were obtained from the San DiegoBlood Bank, San Diego, Calif. The purified PDGF used herein was obtainedfollowing the first two steps of the procedures described in Antoniadeset al., Proc. Natl. Acad. Sci. USA, 76, 1809-1813 (1979).

Briefly, platelets were collected by centrifugation at 28,000×gravity(g) for 20 minutes at 4 degrees C. The obtained platelets were washed byresuspension in 400 milliliters of a mixture containing (a) 9 volumes of17 millimolar Tris-HCl, at pH 7.4 including 0.15 molar NaCl and 1%glucose; and (b) 1 volume of a solution that includes per 100milliliters: 0.8 grams citric acid monohydrate, 2.2 grams anhydrousdextrose and 2.6 grams of sodium citrate dihydrate, followed by furthercentrifugation at 28,000×g for 10 minutes at 4 degrees C. The thuswashed platelets were then resuspended in 16 milliliters of an aqueoussolution containing 0.008 molar NaCl and 0.01 molar phosphate ion at pH7.4 (NaCl-phosphate ion solution), and boiled for 10 minutes to lyse thecells.

Phenylmethyl sulfonyl fluoride and Traysylol (Sigma Chemical Co., St.Louis, Mo.), protease inhibitors, were added to the lysed cells atconcentrations of 1 millimolar and 3%, respectively. The lysed cellmixture was again centrifuged to provide a pellet and a supernatant.

The supernatant was mixed with 8 milliliters of CM Sephadex C-50(Pharmacia Fine Chemicals, Piscataway, N.J.) beads that were previouslyequilibrated in the NaCl-phosphate ion solution. The beads and liquidwere poured into a chromatography column (15×1.5 centimeters) that waswashed with 6 column volumes of the above NaCl-phosphate ion solution.The PDGF, first eluate, was obtained by eluting the column with twocolumn volumes of 1 molar NaCl. Traysylol was added to the eluate toprovide a final concentration of 3%, and the eluate was dialyzed againstthe above NaCl-phosphate ion solution.

The above-produced lysed cell pellet was extracted with a 1 molar NaClsolution for 24 hours at 4 degrees C., and centrifuged. The supernatantwas dialyzed against the above NaCl-phosphate ion solution, admixed withthe above Sephadex, and made into a column. The column was washed andeluted as above to provide a second eluate that was dialyzed as above.The pellet prepared in this procedure was treated the same way toprovide a third eluate that was again dialyzed as discussed before.

The three dialyzed eluates were pooled and concentrated to a fewmilliliters of volume using an Amicon ultrafiltration apparatus (Amicon,Lexington, Mass.) and a filter having a 10 K dakton exclusion. The PDGFso purified was then treated as discussed for FIG. 5.

Purified PDGF extract from approximately 2.5 units of platelets weremixed with a minimal volume of solution containing 0.5 percent sodiumdodecyl sulfate (SDS) and 5 percent of 2-mercaptoethanol. The resultingmixture was boiled for two minutes and then electrophoresed therethrougha 5-17 percent polyacrylamide gel. The protein was thereafterelectrophoretically transferred to nitrocellulose. (Niman and Elder,supra.) that was thereafter cut into strips, following the Western blotprocedure.

The nitrocellulose strips so prepared were then treated with a solutioncontaining 3 percent bovine serum albumin (BSA), 0.1 percentpolyoxyethylene-9-octyl phenyl ether (Triton®X-100) in phosphatebuffered saline to inhibit non-specific protein binding. Four mililitersof mouse anti-serum diluted 1:200 were then incubated with thenitrocellulose strips.

After washing three times with a solution of 0.1 percent Triton®X-100 inPBS, the nitrocellulose strips were incubated either with 10⁶ counts perminute of ¹²⁵ I-labeled Staphylococus aureus protein, or a 1:1000dilution of peroxidase-conjugated goat anti-mouse serum (Tago), andagain washed with 0.1 percent Triton®X-100 in PBS. The preoxidaseconjugate was developed with a solution containing 0.0009 percent H₂ O₂,0.0025 percent 3,3¹ -dimethoxybenzidine dihydrochloride (Eastman-Kodak,Co.,) in a 10 millimolar Tris buffer having a pH value of 7.4. The ¹²⁵ Ilabeled strips were developed by exposure on XRP-1 film (Eastman-KodakCo.) using Cronex Hi-Plus (E.I. DuPont de Nemours & Co.) intensifyingscreens at minus 70° C. for 48 hours.

H. Urine Assay

Urine from donors (patients) as noted in the description of the Figureswas collected and used as collected or concentrated to 40-fold using anAmicon ultrafiltration apparatus. This fluid was employed as the bodyfluid sample aliquot in the assay for proteins encoded by or related tosis, fes and ras oncogenes.

The concentrated urine sample was prepared in the following manner. Theurine was clarified at 6000 r.p.m. at 4° C. for 10 minutes. Thesupernatant was then concentrated using an Amicon filter having a 10,000dalton exclusion. This concentrated urine was then dializedi to separateprotein fractions.

Concentrated urine was electrophoresed at 25 microliters per lane into a5-17% polyacrylamide gel to provide the equivalent of protein from oneml of collected urine, and then electrophoresed onto nitrocellulose. Thenitrocellulose filter was then probed with a 1/200 dilution of, forexample, mouse antiserum in a solution 3% bovine serum albumin, 0.1%Triton® X100 and PBS. The nitrocellulose filter was then washed threetimes and incubated with 10⁶ cpm of ²⁵ I-labeled protein A.

Binding was visualized with intensifying screens at -700 Centigrade asdescribed in FIG. 6, supra.

I. oncoproteins and Transformed Cells

NRK and SSV-transformed NRK cells were provided by S. A. Aaronson and K.C. Robbins of the Center for Cancer Research, National Institutes ofHealth, Bathesda, Md. The cells were grown in Dulbecco's minimalessential medium supplemented with 10% fetal calf serum, 2 millimolarL-glutamine, 100 IU per milliliter of penicillin and 100 micrograms permilliliter of streptomycin.

Parallel cultures of NRK and SSV-transformed NRK cells were washed 3times for 2 hour intervals, and were then incubated for 18 hours inmedium without serum at 15 milliliters per T75 centimeter² flask. Themedium so conditioned was then centrifuged, and was stored frozen at-70° C.

The conditioned medium was thawed, concentrated 500-fold using dialysisin 1 molar acetic acid and was thereafter lyophilized. Aftersolubilization and reduction with 10% 2-mercaptoethanol, 50 microlitersof concentrated, conditioned media were electrophoresed into a 5-17%sodium dodecyl sulfatepolyacrylamide gel. Secreted proteins were thenelectrophoretically transferred and bound to nitrocellulose. Nonspecificbinding was blocked by preincubation of the cell extract wth a solutioncontaining 3% bovine serum albumin and 0.1% polyoxyethylene octyl phenylether in phosphate-buffered saline at a pH value of 7.4.

Prior to carrying out the immunological assays, 20 microliters of mouseantisera induced by PDGF-2(1-17) or PDGF-2(73-89) (described before)were preincubated with 100 micrograms of an appropriate polypeptide for1 hour at 37° C. The oligoclonal antibody-containing/polypeptidereaction mixture was then diluted 1:500 with the above preincubationsolution. The diluted solution so prepared was then contacted at 4° C.with the nitrocellulose-bound conditioned media, and that contact wasmaintained (incubated) for a time period of 15 minutes, a timesufficient for the immunoreaction of the antibody (receptor) and proteinbound on the nitrocellulose. The nitrocellulose was thereafter washed.

The washed nitrocellulose was then contacted with affinity-purifiedrabbit anti-mouse IgG₁ antibodies (Litton).diluted 1:500 at 25° C. Thecontact was maintained for a time period of 2 hours sufficient for theanti-mouse IgG₁ anibodies to immunoreact with antibodies from theantisera that had bound to the nitrocellulose-bound secreted proteins ofthe conditioned media. The nitrocellulose was then washed again.

Immunoreaction (binding) was visualized with 10⁶ counts per minute of¹²⁵ I-labeled Staphylococcus aureus protein A as described in Niman,Nature, 307, 180-183 (1984).

The United States Government has rights in this invention pursuant toPublic Health Service Contract N01-CP-41009, Public Health ServiceGrants CA 38160 and CA25803.

The foregoing is intended as illustrative of the present invention butnot limiting. Numerous variations and modifications may be effectedwithout departing from the true spirit and scope of the novel conceptsof the invention.

What is claimed is:
 1. A method of obtaining proteins in purified form comprising the steps of(a) raising a receptor molecule to a polypeptide consisting of about 7 to about 40 amino acid residues corresponding to a portion of a protein, wherein said receptor molecule immunoreacts with both said protein and said polypeptide; (b) providing an affinity sorbant comprising an inert, solid support having linked thereto said receptor molecule, wherein said affinity-sorbant forms a reversible receptor-ligand complex when admixed with an aqueous composition containing said protein; (c) providing an aqueous composition containing said protein; (d) admixing said affinity sorbant and said aqueous composition to form a reversible, linked receptor-ligand complex between said receptor and said protein of step (a); (e) separating the linked receptor-ligand complex from said aqueous composition to thereby obtain said protein in purified form linked to said affinity sorbant; (f) dissociating said receptor-ligand complex; and (g) separating said purified protein from said affinity sorbant to provide said purified protein free from said affinity sorbant.
 2. The method of claim 1, wherein said protein is an oncoprotein.
 3. The method of claim 1, wherein said polypeptide comprises an amino acid residue sequence, written from left to right and in the direction from amino-terminus to carboxy-terminus, represented by a formula selected from the group consisting ofLMRACWQWNPSDRPSF, FMQACWALEPTRRTF, and AMEQTWRLDPEERPTF.
 4. A method of assaying a body sample for the presence of an oncoprotein ligand, comprising the steps of:raising a monoclonal receptor molecule of the Ig class to a polypeptide consisting of about 7 to about 40 amino acid residues corresponding to a portion of a protein ligand encoded by a retrovirus gene, wherein said monoclonal receptor molecule immunoreacts with both said protein ligand and said polypeptide; admixing said body sample in an aqueous composition with said monoclonal receptor molecule; maintaining said admixture for a time period sufficient for said monoclonal receptor molecule to react with an oncoprotein ligand and form a complex; and determining the presence of said complex.
 5. The method of claim 4 wherein said polypeptide to which said receptor binds comprises an amino acid residue sequence, from left to right in the direction from amino-terminus to carboxy-terminus, represented by a formula selected from the group consisting of:SDVWSFGILLWETFSLGASPYPNLSNQQTR, SPYPNLSNQQTR, VPVKWTAPEALNYG, IHRDLAARNCLVTEKN, SSESDVWSFGILLWE, IGRGNFGEVFSG, LMEQCWAYEPGQRPSF, RRKVEQEGYPQESSKAG, RHYTDEDPEKEKRIKELEL, RKIEIVRKKPIFKKATV, RVTIRTVRVRRPPKGKHRKC, YREQIKRVKDSDDVPMVLVGNKC, KVVVGAR(S,V,G)GVGK, CDEENFYQQQQQSEL, PAPSEDIWKKFEL, LPTPPLSPSRRSGLC, CDPDDETFINKNIIIQDC, CSTSSLYLQDLSAAASEC, CASQDSSAFSPSSIDSLLSSTESSP, LPRELSPSUDSR, RQAASPPHIGGTY, TTREVPYSGEPQ, SLGSLTIAEPAMIAECK, RKIEVIVRKKPIFIKKATV, RVTIRTVRVRRDPKGKHRKC, SIEEAVPAECKT, ENDTLVRKYADANAVCQ, LGSGAFGTIYKG, IMVKCWMIDADSRPKF, LMRACWQWNPSDRPSF, FMQACWALEPTRRTF, LMCQCWRKDPEERPTF, LGQGCFGEVWMG, CGSSKSKPKDPSQRRS and AMEQTWRLDPEERPTFwherein the amino acid residues in parentheses are each an alternative to the immediately preceding amino acid residue in the formula.
 6. The method of claim 4 wherein said body, sample is a cell extract.
 7. The method of claim 4 wherein said body sample is urine.
 8. The method of claim 7 including the further step of concentrating said urine prior to admixture with said receptor.
 9. A method of assaying for the presence of an oncoprotein ligand comprising the steps of:(a) raising a receptor molecule of the Ig class to a polypeptide consisting of about 7 to about 40 amino acid residues corresponding to a portion of an oncoprotein ligand encoded by a retrovirus gene, wherein said receptor molecule immunoreacts with both said oncoprotein and said polypeptide; (b) providing a sample of urine from a donor to be assayed for the presence of said oncoprotein ligand; (c) admixing said sample with said receptor molecule in an aqueous composition; (d) maintaining said admixture for a period of time sufficient for said receptor molecule to react with said oncoprotein ligand and form an immunocomplex; and (e) determining the presence of said immunocomplex.
 10. The method of claim 9 wherein said receptor molecule is oligoclonal.
 11. The method of claim 9 wherein said receptor molecule is monoclonal.
 12. The method of claim 9 wherein said urine is concentrated prior to said admixture with said receptor molecule.
 13. The method of claim 9 wherein said urine sample is affixed to a nitrocellulose matrix as a solid support and said receptor molecule in said aqueous composition is admixed with the affixed urine sample.
 14. The method of claim 9 wherein a plurality of different receptor molecules are provided, each of which is separately admixed with an aliquot of said urine sample, and the presence of an inmiunocomplex is separately determined for each of said admixtures. 